Compositions and methods for improving mitochondrial function and treating neurodegenerative diseases and cognitive disorders

ABSTRACT

Provided are compositions comprising compounds or precursors to compounds which may be used for a variety of therapeutic applications including, for example, treating and/or preventing a disease or disorder related to reduced or inadequate mitochondrial activity, including aging or stress, diabetes, obesity, and neurodegenerative diseases. The compounds relate generally to urolithins and precursors thereof, including but not limited to ellagitannins and urolithin A. In certain embodiments the compositions are presented in or as food products or nutritional supplements. These same compounds and compositions can also be used advantageously in generally healthy individuals to increase or maintain metabolic rate, decrease percent body fat, increase or maintain muscle mass, manage body weight, improve or maintain mental performance (including memory), improve or maintain muscle performance, improve or maintain mood, and manage stress.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/218,790, filed Jul. 25, 2016; which is a divisional of U.S. patentapplication Ser. No. 14/644,912, filed Mar. 11, 2015; which is adivisional of U.S. patent application Ser. No. 13/336,841, filed Dec.23, 2011, now U.S. Pat. No. 9,872,850; which claims benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/426,957,filed Dec. 23, 2010.

BACKGROUND OF THE INVENTION

Ellagitannins are monomeric, oligomeric, and polymeric polyphenols thatare abundant in some fruits, berries and nuts, such as pomegranates,raspberries, strawberries, black raspberries, walnuts and almonds. Thefruits and berries are widely consumed fresh and as beverages, such asjuice, and these have been reported to promote health.

In commercial fruit juice processing methods, ellagitannins, which areparticularly abundant in some fruit peels, are extracted in largequantities into the juice. Ellagitannins belong to the chemical class ofhydrolyzable tannins, which release ellagic acid upon hydrolysis. Invitro studies have suggested that ellagitannins, at concentrations inthe range of 10-100 micromolar (μM), have potential anti-oxidant,anti-atherogenic, anti-thrombotic, anti-inflammatory, andanti-angiogenic effects. Fruits may have different ellagitannins thatare predominant, for example, in fruit juice prepared from pomegranate,the predominant ellagitannin is punicalagin [2,3hexahydroxydiphenoyl-4,6-gallagylglucose], which occurs as a mixture ofisomers. The reported potent anti-oxidant properties of pomegranatejuice have been attributed to the high content of punicalagin isomers,which can reach levels >2 g/L of juice. Ellagitannins have also beenidentified as the active anti-atherogenic compounds in pomegranatejuice. It has also been suggested that pomegranate ellagitannins andpomegranate fruit extracts inhibit the proliferation of human cancercells and modulate inflammatory sub-cellular signaling pathways andapoptosis. See, for example, Seeram et al. (2005) J Nutr Biochem.16:360-7; Adams et al. (2006) J Agric Food Chem. 54:980-85; Afaq et al.(2005) Photochem Photobiol. 81:38-45; Afaq et al. (2005) Int J Cancer.113:423-33. Pomegranate fruit extract has also been reported to reduceprostate tumor growth and prostate serum antigen (PSA) levels in athymicnude mice implanted with CWR22Rv1 prostate cells. Malik et al. (2005)Proc Natl Acad Sci. 102:14813-8.

Unfortunately, for the most part ellagitannins are poorly absorbed bythe human gut. However, a number of metabolites derived fromellagitannins are absorbed by the human gut, including certainmetabolites ultimately formed in the gut by commensal microorganisms(i.e., intestinal microflora).

Ellagitannins release ellagic acid under physiological conditions invivo, and ellagic acid is then gradually metabolized by the gutmicroflora in the intestine to produce urolithin D, urolithn C,urolithin A (UA) and urolithin B (UB). Once the metabolites areabsorbed, they undergo glucuronidation and once in the liver, they arefurther metabolized to produce glucuronides, and/or sulfates, to give acombination of metabolites secreted in the bile.

Urolithins are metabolites of ellagic acid, punicalagin (PA), punicalin(PB), tellimagrandin (TL), and other ellagitannins (Cerda, Espin et al.2004; Cerda, Periago et al. 2005). Ellagic acid (EA) is abundant inpomegranate juice (Gil, Tomas-Barberan et al. 2000). The ellagitannintellimagrandin (TL) has been previously isolated and characterizedbefore from pomegranate and other plants (Tanaka, Nonaka et al. 1986;Tanaka, Nonaka et al. 1986; Satomi, Umemura et al. 1993). Structuralformulas for UA, PA, PB, EA, and TL are presented in FIG. 1.

Considerable efforts have been made to understand the mechanism ofmetabolic disorders, neurodegeneration and cognitive decline, so as tobetter design treatment modalities including those based on naturalproducts. One of the key observations has been therole of decliningmitochondrial energy production, corresponding with increased oxidativestress and apoptosis, plays a significant role in degenerative diseasesand the process of aging. A variety of degenerative diseases have nowbeen shown to be caused by mutations in mitochondrial genes encoded bythe mitochondrial DNA (mtDNA) or the nuclear DNA (nDNA). Importantly,somatic mtDNA mutations accumulate with age in post-mitotic tissues inassociation with the age-related decline in mitochondrial function andare thought to be an important factor in aging and senescence. Inheriteddiseases can result from mtDNA base substitution and rearrangementmutations and can affect the CNS, heart and skeletal muscle, and renal,endocrine and hematological systems.

Mitochondria generate most of the cellular energy by oxidativephosphorylation (OXPHOS), and they produce most of the toxic reactiveoxygen species (ROS) as a by-product. Genetic defects that inhibitOXPHOS also cause the redirection of OXPHOS electrons into ROSproduction, thus increasing oxidative stress. A decline in mitochondrialenergy production and an increase in oxidative stress can impinge on themitochondrial permeability transition pore (mtPTP) to initiateprogrammed cell death (apoptosis). The interaction of these threefactors is believed to play a major role in the pathophysiology ofdegenerative diseases and the aging process, which affects all tissuesof the body.

In the normal brain, optimal cognitive function mainly relies on theactivity and communication between neurons, highly complex cells able toconvey electric signals and elicit chemical neurotransmission. Neuronalfunction depends on long and complex cellular processes that can extendover centimeters or even meters to connect neurons or target cells, andcan make more than 100,000 synaptic contacts. As such, neurons arehighly dependent on energy supply and, therefore, are exposed tooxidative stress damage. Cognitive function is dependent on a carefulbalance of intracellular signaling that takes place within a complexnetwork of neurons. Optimal cognitive function can be impaired bynumerous factors such as aging, cellular stress, chronic stress, andneurodegenerative disorders. Cognitive decline may be characterized by adecrease in performance in thinking, learning, memory, alertness, and/orimpaired psychological skills, as well as by depression and anxiety.

Mitochondrial function has also been shown to be important in metabolicdisorders. Diabetes and obesity have been correlated with compromises inmitochondrial function. It has been suggested that the couplingefficiency in mitochondria, or the proportion of oxygen consumptionnecessary to make ATP, is related to levels of obesity, with highcoupling efficiency possibly resulting in higher deposition of fatstores (Harper, Green et al. 2008). In diabetes, recent work hassuggested that mitochondrial dysfunction is a cause of insulininsensitivity in myocytes and adipocytes, as a result of insufficientenergy supply or defects in the insulin signaling pathway (Wang, Wang etal. 2010).

SUMMARY OF THE INVENTION

The invention relates to compositions comprising compounds or precursorsto compounds which may be used for a variety of therapeutic applicationsincluding, for example, treating and/or preventing disease or disordersrelated to reduced or inadequate mitochondrial activity, including agingor stress, diabetes, obesity, and neurodegenerative diseases. These samecompounds and compositions can also be used advantageously in generallyhealthy individuals to increase or maintain metabolic rate, decreasepercent body fat, increase or maintain muscle mass, manage body weight,improve or maintain mental performance (including memory), improve ormaintain muscle performance, improve or maintain mood, and managestress.

An object of the present invention provides a plant extract, activefraction thereof, or one or more active components or metabolitesisolatable therefrom or synthesized, for use in the prophylaxis ortreatment of a disease state initiated or characterized (i) byinadequate mitochondrial activity; (ii) by metabolic disorders such asdiabetes and obesity; (iii) by a decline in cognitive function; or (iv)by mood disturbances.

Accordingly, in a first aspect, the invention provides a fruit extract,active fraction thereof, or one or more active components isolatabletherefrom, for use as an inducer of mitochondrial function.

As used herein, the term “fraction” refers to purified or partiallypurified extracts.

In another aspect, the invention provides a fruit extract, activefraction thereof, or one or more active components isolatable therefrom,for use in the prophylaxis or treatment of a disease state initiated orcharacterized by reduced mitochondrial function.

In another aspect, the invention provides the use of a fruit or anextract, or active fraction thereof, or one or more active componentsisolatable therefrom, as hereinbefore defined for the manufacture of amedicament for use in (i) the prophylaxis or treatment of a diseasestate initiated or characterized by reduced mitochondrial function; or(ii) improving cognitive or muscular function. Such disease states caninclude, without limitation, neurodegenerative disease, cognitivedisorder, mood disorder, anxiety disorder, metabolic disorder, diabetesmellitus, and obesity.

In another aspect, the invention provides a process for the manufactureof a medicament for use in (i) the prophylaxis or treatment of a diseasestate initiated or characterized by reduced mitochondrial function; or(ii) improving cognitive or muscular function; which process ischaracterized by the use, as an essential ingredient of the medicament,of a fruit, or an extract or active fraction thereof or one or moreactive components isolatable therefrom as hereinbefore defined.

In a still further aspect, the invention provides a pharmaceuticalcomposition comprising an active component derived from a fruit or anextract or active fraction or one or more active components isolatabletherefrom as hereinbefore defined and a pharmaceutically acceptablecarrier.

An object of the present invention is to provide plant extracts, activefraction thereof, or one or more active components or metabolitesisolatable therefrom, or synthesized, for use in treating diseases ordisorders in a subject that would benefit from increased mitochondrialactivity, for improving (i) brain function, (ii) metabolic function,including diabetes or obesity, (iii) muscle performance and (iv)increasing tissue ATP levels.

An object of the present invention is to provide extracts, compositionsand compounds which are neuroprotective, neurotrophic, and/or promoteneurite outgrowth and, consequently, improve cognitive function, as wellas methods of use of these compounds and compositions.

An object of the present invention is to provide compounds andcompositions that improve, protect, and maintain brain function andcognition. Another object of this invention is to improve, protectagainst and manage mood disorders. Another object of this invention isto protect against stress-induced or stress-associated disorders orsymptoms.

An object of this invention is to provide neuroprotective compounds toprotect the brain from insults, as well as improve cognitive performanceand memory in normal adults. Another object of the present invention isto provide new compounds that stimulate neuronal plasticity. Neuronalplasticity is well known to be a key process necessary for memory andcognitive functions. Such compounds may influence neurite outgrowth,number of branches per cells, mean processes per cells and even numbersof synapses formed.

The invention also relates to several polyphenol compounds andderivatives thereof, related to ellagitannins, as bioactive naturalcompounds found in pomegranate and other fruit, as well as bioactivenatural extracts which contain these compounds. These compounds includeellagitannins, punicalagin, and ellagic acid, all which are found in thepomegranate, but can also be isolated from other fruits and berries, aswell as metabolites of these compounds. As disclosed herein, thesecompounds have now been shown to have beneficial effects on (i)mitochondrial function, (ii) cellular metabolism, and (iii) neuronalplasticity.

Using in vitro modeling of neurite outgrowth and process formation inneuronal cell culture and primary cells, various compounds were examinedfor their beneficial effects. As described above, aging,neurodegeneration, and chronic stress have negative impacts on neuriteoutgrowth. Remarkably, it has been discovered that the compounds of thepresent invention have neuroprotective properties, exhibit strongstimulatory activity in PC-12 cells and primary mesencephalic neurons,and improve cognitive function and memory in animal models.

In one aspect, the invention relates to a composition, such as apharmaceutical, a medical food, a functional food, a food additive, or adietary supplement, comprising the compounds or a mixture thereof of theinvention. The composition may also optionally contain an additionaltherapeutic agent, or may be administered in combination with anothertherapeutic compound. Packaged products, containing the above-mentionedcomposition and a label and/or instructions for use in improving memoryand cognitive performance and or for the treatment of a disease orcondition associated with damage to the brain typical for conditionsfound in the aging adult, are also provided.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of pomegranate extract: for the treatmentor prevention of a condition selected from the group consisting ofobesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of an ellagitannin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of punicalagin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of ellagic acid: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of a urolithin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

In each of the foregoing, in one embodiment the condition is obesity.

In each of the foregoing, in one embodiment the condition is reducedmetabolic rate.

In each of the foregoing, in one embodiment the condition is metabolicsyndrome.

In each of the foregoing, in one embodiment the condition is diabetesmellitus.

In each of the foregoing, in one embodiment the condition iscardiovascular disease.

In each of the foregoing, in one embodiment the condition ishyperlipidemia.

In each of the foregoing, in one embodiment the condition isneurodegenerative disease.

In each of the foregoing, in one embodiment the condition is cognitivedisorder.

In each of the foregoing, in one embodiment the condition is mooddisorder.

In each of the foregoing, in one embodiment the condition is stress.

In each of the foregoing, in one embodiment the condition is anxietydisorder.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for weight management.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for increasing muscle performance.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for increasing mental performance.

An aspect of the invention is a method of increasing or maintainingmitochondrial function. The method includes the step of contacting cellswith an effective amount of a urolithin or a precursor thereof, toincrease function of the mitochondria.

An aspect of the invention is a method of treating, preventing, ormanaging a mitochondria-related disease or condition associated with analtered mitochondrial function or a reduced mitochondrial density. Themethod includes the step of administering to a subject in need thereof atherapeutically effective amount of a urolithin or a precursor thereof,to treat the disease or condition associated with altered mitochondrialfunction or reduced mitochondrial density.

An aspect of the invention is a method of increasing metabolic rate. Themethod includes the step of administering to a subject in need thereofan effective amount of a urolithin or a precursor thereof, to increasemetabolic rate.

An aspect of the invention is a method of preventing or treatingmetabolic syndrome. The method includes the step of administering to asubject in need thereof an effective amount of a urolithin or aprecursor thereof, to prevent or treat metabolic syndrome.

An aspect of the invention is a method of preventing or treatingobesity. The method includes the step of administering to a subject inneed thereof an effective amount of a urolithin or a precursor thereof,to prevent or treat obesity.

An aspect of the invention is a method of preventing or treatingcardiovascular disease. The method includes the step of administering toa subject in need thereof an effective amount of a urolithin or aprecursor thereof, to prevent or treat cardiovascular disease.

An aspect of the invention is a method of treating hyperlipidemia. Themethod includes the step of administering to a subject in need thereofan effective amount of a urolithin or a precursor thereof, to treathyperlipidemia. In one embodiment, the hyperlipidemia ishypertriglyceridemia. In one embodiment, the hyperlipidemia is elevatedfree fatty acids.

An aspect of the invention is a method of treating a metabolic disorder.The method includes the step of administering to a subject in needthereof a therapeutically effective amount of a urolithin or a precursorthereof, to treat the metabolic disorder. In one embodiment, themetabolic disorder is diabetes mellitus. In one embodiment, themetabolic disorder is obesity.

An aspect of the invention is a method of treating a neurodegenerativedisease. The method includes the step of administering to a subject inneed thereof a therapeutically effective amount of a urolithin or aprecursor thereof, to treat the neurodegenerative disease. In oneembodiment, the neurodegenerative disease is selected from the groupconsisting of AIDS dementia complex, Alzheimer's disease, amyotrophiclateral sclerosis, adrenoleukodystrophy, Alexander disease, Alper'sdisease, ataxia telangiectasia, Batten disease, bovine spongiformencephalopathy (BSE), Canavan disease, corticobasal degeneration,Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familialinsomnia, frontotemporal lobar degeneration, Huntington's disease,Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease,multiple sclerosis, multiple system atrophy, neuroacanthocytosis,Niemann-Pick disease, Parkinson's disease, Pick's disease, primarylateral sclerosis, progressive supranuclear palsy, Refsum disease,Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellarataxia, subacute combined degeneration of spinal cord, tabes dorsalis,Tay-Sachs disease, toxic encephalopathy, transmissible spongiformencephalopathy, and wobbly hedgehog syndrome. In one embodiment, theneurodegenerative disease is selected from the group consisting ofAlzheimer's disease, amyotrophic lateral sclerosis, Huntington'sdisease, and Parkinson's disease. In one embodiment, theneurodegenerative disease is Alzheimer's disease.

An aspect of the invention is a method of improving cognitive function.The method includes the step of administering to a subject in needthereof an effective amount of a urolithin or a precursor thereof, toimprove cognitive function. In one embodiment, the cognitive function isselected from the group consisting of perception, memory, attention,speech comprehension, speech generation, reading comprehension, creationof imagery, learning, and reasoning. In one embodiment, the cognitivefunction is selected from the group consisting of perception, memory,attention, and reasoning. In one embodiment, the cognitive function ismemory.

An aspect of the invention is a method of treating a cognitive disorder.The method includes the step of administering to a subject in needthereof a therapeutically effective amount of a urolithin or a precursorthereof, to treat the cognitive disorder. In one embodiment, thecognitive disorder is selected from the group consisting of delirium,dementia, learning disorder, attention deficit disorder (ADD), andattention deficit hyperactivity disorder (ADHD). In one embodiment, thecognitive disorder is a learning disorder. In one embodiment, thecognitive disorder is attention deficit disorder (ADD). In oneembodiment, the cognitive disorder is attention deficit hyperactivitydisorder (ADHD).

An aspect of the invention is a method of treating stress-induced orstress-related cognitive deficit. The method includes the step ofadministering to a subject in need thereof a therapeutically effectiveamount of a urolithin or a precursor thereof, to treat thestress-induced or stress-related deficit.

An aspect of the invention is a method of treating a mood disorder. Themethod includes the step of administering to a subject in need thereof atherapeutically effective amount of a urolithin or a precursor thereof,to treat the mood disorder. In one embodiment, the mood disorder isselected from the group consisting of depression, postpartum depression,dysthymia, and bipolar disorder. In one embodiment, the mood disorder isdepression. In one embodiment, the mood disorder is dysthymia.

An aspect of the invention is a method of treating stress-induced orstress-related mood disorder, e.g., dysthymia. The method includes thestep of administering to a subject in need thereof a therapeuticallyeffective amount of a urolithin or a precursor thereof, to treat thestress-induced or stress-related mood disorder.

An aspect of the invention is a method of treating an anxiety disorder.The method includes the step of administering to a subject in needthereof a therapeutically effective amount of a urolithin or a precursorthereof, to treat the anxiety disorder. In one embodiment, the anxietydisorder is selected from the group consisting of generalized anxietydisorder, panic disorder, panic disorder with agoraphobia, agoraphobia,social anxiety disorder, obsessive-compulsive disorder, andpost-traumatic stress disorder. In one embodiment, the anxiety disorderis generalized anxiety disorder. In one embodiment, the anxiety disorderis post-traumatic stress disorder.

An aspect of the invention is a method of treating stress-induced orstress-related anxiety. The method includes the step of administering toa subject in need thereof a therapeutically effective amount of aurolithin or a precursor thereof, to treat the stress-induced orstress-related anxiety.

An aspect of the invention is a method of enhancing muscle performance.The method includes the step of administering to a subject in needthereof a therapeutically effective amount of a urolithin or a precursorthereof, to increase muscle performance. In one embodiment, the muscleperformance is selected from the group consisting of strength, speed,and endurance.

An aspect of the invention is a method of treating a muscle orneuromuscular disease. The method includes the step of administering toa subject in need thereof a therapeutically effective amount of aurolithin or a precursor thereof, to treat the muscle or neuromusculardisease. In one embodiment, the muscle or neuromuscular disease is amyopathy. In one embodiment, the muscle or neuromuscular disease is amuscular dystrophy. In one embodiment, the muscle or neuromusculardisease is Duchenne muscular dystrophy.

An aspect of the invention is a method of promoting neurite outgrowth.The method includes the step of contacting a nerve cell with aneffective amount of a urolithin or a precursor thereof, to promoteneurite outgrowth. In one embodiment, the contacting comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the urolithin or precursor thereof, to promote neuriteoutgrowth.

The following embodiments can pertain to each aspect and embodiment ofthe invention described and, as appropriate, to each other.

In one embodiment, the urolithin or precursor thereof is an isolatedurolithin.

In one embodiment, the urolithin or precursor thereof is an isolatedurolithin precursor.

In one embodiment, the urolithin is selected from the group consistingof urolithin A, urolithin B, urolithin C, urolithin D, as well as theirmetabolites, including, by means of example, their glucuronidated,methylated, and sulfated forms, and combinations of these urolithins.

In one embodiment, the urolithin or precursor thereof is administered asa natural food selected from the group consisting of berries, grapes,pomegranates, rose hips, and nuts.

In one embodiment, the urolithin or precursor thereof is administered asa processed food product, including as means of example a juice,concentrate, or extract, based on a natural food selected from the groupconsisting of berries, grapes, pomegranates, rose hips, and nuts.

In one embodiment, the urolithin or precursor thereof is administered aspomegranate juice, concentrate, or extract.

In one embodiment, the urolithin or precursor thereof is administered asan ellagitannin.

In one embodiment, the urolithin or precursor thereof is administered aspunicalagin.

In one embodiment, the urolithin or precursor thereof is administered asellagic acid.

In one embodiment, the urolithin or precursor thereof is administered asa urolithin.

In one embodiment, the urolithin or precursor thereof is administeredorally.

In one embodiment, the urolithin or precursor thereof is administeredparenterally.

In one embodiment, the urolithin or precursor thereof is administered atleast weekly. In various embodiments, the urolithin or precursor thereofis administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 times weekly.

In one embodiment, the urolithin or precursor thereof is administered atleast daily. In various embodiments, the urolithin or precursor thereofis administered 1, 2, 3, 4, 5, 6, 7, or 8 times daily.

In one embodiment, the urolithin or precursor thereof is administered ina dose equal or equivalent to 0.1-150 milligram (mg) of urolithin perkilogram (kg) body weight. In one embodiment, the urolithin or precursorthereof is administered in a dose equal or equivalent to 2-120 mg ofurolithin per kg body weight. In one embodiment, the urolithin orprecursor thereof is administered in a dose equal or equivalent to 4-90mg of urolithin per kg body weight. In one embodiment, the urolithin orprecursor thereof is administered in a dose equal or equivalent to 8-30mg of urolithin per kg body weight.

In one embodiment, the urolithin or precursor thereof is administered ina dose sufficient to achieve a peak serum level of at least 0.001micromolar (μM). In one embodiment, the urolithin or precursor thereofis administered in a dose sufficient to achieve a peak serum level of atleast 0.01 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 0.1 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 1 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 10 μM.

In one embodiment, the urolithin or precursor thereof is administered ina dose sufficient to achieve a sustained serum level of at least 0.001micromolar (μM). In one embodiment, the urolithin or precursor thereofis administered in a dose sufficient to achieve a sustained serum levelof at least 0.01 μM. In one embodiment, the urolithin or precursorthereof is administered in a dose sufficient to achieve a sustainedserum level of at least 0.1 μM. In one embodiment, the urolithin orprecursor thereof is administered in a dose sufficient to achieve asustained serum level of at least 1 μM. In one embodiment, the urolithinor precursor thereof is administered in a dose sufficient to achieve asustained serum level of at least 10 μM.

In one embodiment, the subject is not receiving a urolithin or aprecursor thereof, to treat another condition calling for administrationof a urolithin or a precursor or metabolite thereof, selected from thegroup consisting of atherosclerosis, thrombosis, cancer, unwantedangiogenesis, infection, and inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts structural formulas for urolithin A (UA), ellagic acid(EA), tellimagrandin (TL), punicalagin (PA), and punicalin (PB).

FIG. 2 depicts ellagic acid (EA) and its metabolites, urolithin D (UD),urolithin C (UC), urolithin A (UA), and urolithin B (UB), which areproduced by intestinal microflora in animals, including humans.

FIG. 3 is a pair of bar graphs depicting mitochondrial gene expressionlevels in response to the indicated concentrations of ellagic acid(upper panel) and urolithin A (lower panel).

FIG. 4 is a bar graph depicting citrate synthase (CS) activity measuredin vitro in the presence of the indicated concentrations of punicalagin,ellagic acid, urolithin A, or negative control.

FIG. 5A is a collage of immunoblots (IB) depicting effects of ellagicacid (EA) and urolithin A (UA) at the indicated concentrations on levelsof AMP-Activated Protein Kinase (AMPK) and activated, phosphorylatedAMPK (P-AMPK). P-AMPK: phosphorylated AMPK. Control: negative control;RSV: resveratrol positive control.

FIG. 5B is a bar graph depicting densitometric analysis of bands in FIG.5A showing the relative level of activated P-AMPK following treatmentsas compared to control treated cells.

FIG. 6 is a bar graph depicting the total cell numbers for cultures ofPC-12 cells following treatment with 0.5 μM of the indicated compounds.PA, punicalagin; PB, punicalin; UA, urolithin A; EA, ellagic acid; T1,tellimagrandin.

FIG. 7 is a bar graph depicting the mean neurite outgrowth (μm) in PC-12cells following treatment with 0.5 μM of the indicated compounds.Outgrowth is expressed per cell. SP, SP600125; dbcAMP, dibutyryl cyclicAMP; PA, punicalagin; PB, punicalin; UA, urolithin A; EA, ellagic acid;T1, tellimagrandin.

FIG. 8 is a bar graph depicting the percentage of PC-12 cells showingextensive neurite outgrowth (>20 μm) following treatment with 0.5 μM ofthe indicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP; PA,punicalagin; PB, punicalin; UA, urolithin A; EA, ellagic acid; T1,tellimagrandin.

FIG. 9 is a bar graph depicting the mean process formation in PC-12cells following treatment with 0.5 μM of the indicated compounds. SP,SP600125; dbcAMP, dibutyryl cyclic AMP; PA, punicalagin; PB, punicalin;UA, urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIG. 10 is a bar graph depicting the mean outgrowth per cell of primarydopaminergic tyrosine hydroxylase (TH)-positive neurons followingtreatment with 0.1 μM of the indicated compounds. SP, SP600125; dbcAMP,dibutyryl cyclic AMP; UA, urolithin A; EA, ellagic acid; T1,tellimagrandin.

FIG. 11 is a bar graph depicting the percentage of primary dopaminergicTH-positive neurons showing extensive neurite outgrowth (>20 μm)following treatment with 0.1 μM of the indicated compounds. SP,SP600125; dbcAMP, dibutyryl cyclic AMP; UA, urolithin A; EA, ellagicacid; T1, tellimagrandin.

FIG. 12 is a bar graph depicting the mean number of processes formed inprimary dopaminergic TH-positive neurons following treatment with 0.1 μMof the indicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP;UA, urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIG. 13 is a bar graph depicting the maximum process length in primarydopaminergic TH-positive neurons following treatment with 0.1 μM of theindicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP; UA,urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIG. 14 is a bar graph depicting the mean branches per primarydopaminergic TH-positive neuron following treatment with 0.1 μM of theindicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP; UA,urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIG. 15 is a bar graph depicting the mean number of dendrites perprimary dopaminergic TH-positive neuron following treatment with 0.1 μMof the indicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP;UA, urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIG. 16 is a bar graph depicting the mean dendrite length per primarydopaminergic TH-positive neuron following treatment with 0.1 μM of theindicated compounds. SP, SP600125; dbcAMP, dibutyryl cyclic AMP; UA,urolithin A; EA, ellagic acid; T1, tellimagrandin.

FIGS. 17A-C are three series of bar graphs depicting effects ofurolithin A, punicalagin, and pomegranate extract (PE) treatment on theonset of obesity in high fat diet (HFD)-fed mice. Urolithin A wasadministered as food admix; PE and punicalagin were administered bygavage. FIG. 17A depicts body weight follow-up expressed as percentageincrease compared to initial body weight. FIG. 17B depicts percentagefat mass measured by EchoMRI after 5 weeks of treatment. FIG. 17Cdepicts percentage lean mass measured by EchoMRI after 5 weeks oftreatment. Group composition: HFD control (food admix): n=10; HFDcontrol (gavage): n=10; HFD plus urolithin A (food admix): n=9; HFD pluspunicalagin (gavage): n=8; HFD plus PE (gavage): n=7. Results areexpressed as mean±SEM. * p<0.05 (Student's t-test). For panel A, resultswere analyzed by 2-way ANOVA. Values of p are indicated.

FIGS. 18A and B are two pairs of bar graphs depicting effects of ellagicacid and urolithin A on lean mass and fat mass in mice fed standard chowdiet. FIG. 18A depicts the percentage of lean mass (muscle) measured byEchoMRI after 2 weeks of treatment.

FIG. 18B depicts the percentage of fat mass (muscle) measured by EchoMRIafter 2 weeks of treatment. Group composition: Chow diet control (foodadmix): n=8; Chow diet plus ellagic acid (food admix): n=7; Chow dietplus urolithin A (food admix): n=7. Results are expressed as mean±SEM. *p<0.05 (Student's t-test).

FIGS. 19A and 19B is a pair of graphs and a corresponding pair of bargraphs depicting effects of ellagic acid and urolithin A on oxygenconsumption in mice fed standard chow diet. FIG. 19A depicts thefollow-up of oxygen consumption over a 20 h period. Filled barscorrespond to the dark phase (7 pm to 7 am). The rest corresponds to thelight phase. FIG. 19B depicts the oxygen consumption represented as thearea under the curve (AUC). Group composition: Chow diet control (foodadmix): n=8; Chow diet plus ellagic acid (food admix): n=7; Chow dietplus urolithin A (food admix): n=7. Results are expressed as mean±SEM. *p<0.05 (Student's t-test). For panel A, results were analyzed by 2-wayANOVA. Value of p is indicated (Chow diet control vs Chow diet plustreatment).

FIGS. 20A and 20B are a series of graphs and a corresponding series ofbar graphs depicting effect of urolithin A, punicalagin, and pomegranateextract (PE) on oxygen consumption in mice fed a high-fat diet (HFD).FIG. 20A depicts the follow-up of oxygen consumption over a 20 h period.Filled bars correspond to the dark phase (7 pm to 7 am). The restcorresponds to the light phase. FIG. 20B depicts the oxygen consumptionrepresented as the area under the curve (AUC). Group composition: HFDcontrol (food admix): n=10; HFD control (gavage): n=10; HFD plusurolithin A (food admix): n=9; HFD plus punicalagin (gavage): n=8; HFDplus PE (gavage): n=7. Results are expressed as mean±SEM. * p<0.05(Student's t-test). For panel A, results were analyzed by 2-way ANOVA.

FIGS. 21A and 21B is a pair of graphs and a corresponding pair of bargraphs depicting effect of ellagic acid and urolithin A on respiratoryexchange ratio (RER) in mice fed standard chow diet. FIG. 21A depictsthe follow-up of RER over a 20 h period. Filled bars correspond to thedark phase (7 pm to 7 am). The rest corresponds to the light phase. FIG.21B depicts the RER represented as mean RER. Group composition: Chowdiet control (food admix): n=8; Chow diet plus ellagic acid (foodadmix): n=7; Chow diet plus urolithin A (food admix): n=7. Results areexpressed as mean±SEM. * p<0.05 (Student's t-test). For panel A, resultswere analyzed by 2-way ANOVA. Value of p is indicated (Chow diet controlvs Chow diet plus treatment).

FIGS. 22A and 22B are a series of graphs and a corresponding series ofbar graphs depicting effect of urolithin A, punicalagin, and pomegranateextract (PE) on respiratory exchange ratio (RER) in mice fed a high-fatdiet (HFD). FIG. 22A depicts the follow-up of RER over a 20 h period.FIG. 22B depicts the RER represented as the mean RER. Group composition:HFD control (food admix): n=10; HFD plus urolithin A (food admix): n=9;HFD plus punicalagin (food admix): n=10; HFD plus PE (food admix): n=10.Results are expressed as mean±SEM. * p<0.05 (Student's t-test). Forpanel A, results were analyzed by 2-way ANOVA.

FIGS. 23A and 23B are two series of graphs depicting effect of urolithinA, punicalagin, and pomegranate extract (PE) on triglycerides and freefatty acids in mice fed a high-fat diet (HFD). FIG. 23A depicts theplasma levels of triglycerides in HFD-fed mice treated for 14 weeks.FIG. 23B depicts the plasma levels of free fatty acids in HFD-fed micetreated for 14 weeks. Group composition: HFD control (food admix): n=10;HFD control (gavage): n=10; HFD plus urolithin A (food admix): n=9; HFDplus punicalagin (gavage): n=8; HFD plus PE (gavage): n=7. Results areexpressed as mean±SEM. * p<0.05 (Student's t-test).

FIGS. 24A-C is a series of graphs depicting effect of urolithin A,ellagic acid, and punicalagin on glycemia in mice fed a high-fat diet(HFD). FIG. 24A depicts the glucose tolerance test in HFD-fed micetreated by food admix with urolithin A for 10 weeks.

FIG. 24B depicts the glucose tolerance test in HFD-fed mice treated byfood admix with ellagic acid for 10 weeks. FIG. 24C depicts the glucosetolerance test in HFD-fed mice treated by food admix with punicalaginfor 10 weeks. Group composition: HFD control (food admix): n=10; HFDplus urolithin A (food admix): n=9; HFD plus punicalagin (food admix):n=10. Results are expressed as mean±SEM. * p<0.05 (Student's t-test).

FIGS. 25A and 25B are a line graph and a bar graph depicting effect ofurolithin A (UA) on basal and uncoupled respiration (oxygen consumption)in old (10-day-old) C. elegans. FIG. 25A depicts the basal and uncoupledrespiration (FCCP) in 10-day-old control worms treated with 0.1% DMSOand 10 day-old-worms treated with 30 μM urolithin A in 0.1% DMSO. FIG.25B depicts the representative area under the curve (AUC) of uncoupled(FCCP) respiration in 10-day-old control worms treated with vehicle(0.1% DMSO) or 30 μM urolithin A in 0.1% DMSO. Results are expressed asmean±SEM. * p<0.05 (Student's t-test). OCR, oxygen consumption rate.

FIG. 26 is a bar graph depicting the effect of urolithin A onmitochondria in muscle of C. elegans. Transgenic C. elegans strainSJ4103 shows fluorescence due to muscle-specific expression of greenfluorescent protein (GFP) which is targeted to the mitochondriamembrane. Mitochondria presence in the muscle of the C. elegans is shownby an increase in fluorescence. Results are expressed as mean±SEM. *p=0.0014 (Student's t-test).

FIG. 27 is a bar graph depicting the mobility of mice subjected tochronic stress with or without treatment with pomegranate extract.

FIG. 28 is a bar graph depicting the extent of a “freezing” response ofmice in an anxiety-inducing context with or without treatment withpomegranate extract.

FIG. 29 is a bar graph depicting the effect on mice of administration ofpomegranate extract on the extent of anxiety-induced inhibition ofrearing.

FIG. 30 is a bar graph depicting the effect of the administration ofpomegranate extract on the extent of anxiety-induced inhibition ofgrooming in mice.

FIG. 31 is a line graph depicting the extinction of a memory to aparticular adverse context when repeatedly exposed to the context in theabsence of the adverse effect. Data is shown for mice that haveundergone early-life stress, normally reared control mice, and mice thathave undergone early-life stress but are treated with the ellagitanninpunicalagin. Freezing (%) is expressed as a percentage of the freezingtime during the initial exposure to the context.

FIG. 32 is a graph depicting the effect on mice of chronic stress oneffective learning in the Morris water maze.

FIG. 33 is a bar graph depicting the effect on chronically stressed miceof administration of pomegranate extract on learning performance in theMorris water maze.

FIG. 34 is a graph depicting the accumulated distance from a hiddenplatform for several trials during the training phase in the Morriswater maze, a measurement of cognitive learning. Data is shown for micethat have undergone early-life stress, normally reared control mice, andmice that have undergone early-life stress but are treated with theellagitannin punicalagin. Distance to platform is the sum of accumulateddistances between the mouse and the hidden platform for all intervalsmeasured (25 intervals/sec) during the observation period (60 sec).

FIG. 35 is a bar graph depicting memory of aged rats in a socialrecognition test when treated with either the pomegranate extract 1108or a control (Ctrl).

FIG. 36 is a bar graph depicting Morris water maze results for aged ratstreated with pomegranate extract 1108 or control (Ctrl).

FIG. 37 is a bar graph depicting the percent of correct alterations in aY maze for the Alzheimer disease mouse model SXFAD, both treated anduntreated, as well as normal control mice. Significance: **p<0.01,*p<0.05, one way ANOVA.

FIG. 38 is a bar graph depicting Morris water maze results fortransgenic mice modeling Alzheimer's disease (hAPP-Tg) treated withpomegranate-derived extracts 31008, 61109, 71109, or control (Vehicle).Also shown are results for wild-type mice (Non-Tg) treated with control(Vehicle).

FIG. 39 is a bar graph depicting dark/light box results for mice thathave undergone early-life stress versus normally reared control mice,and mice having undergone early-life stress and treated with theellagitannin punicalagin. Results are expressed as mean±SEM.Significance: *p<0.05, (Student's t-test).

FIG. 40 is a bar graph depicting results for the elevated 0-maze formice that have undergone early-life stress versus normally rearedcontrol mice, and mice having undergone early-life stress and treatedwith the ellagitannin punicalagin. Results are expressed as mean±SEM.Significance: *p<0.05, (Student's t-test).

FIG. 41 is a bar graph depicting results for the forced swim test formice that have undergone early-life stress versus normally rearedcontrol mice, and mice having undergone early-life stress and treatedwith the ellagitannin punicalagin. Results are expressed as mean±SEM.Significance: *p<0.05, **p<0.01 (Student's t-test).

FIG. 42 is a bar graph depicting results for the training in thecontextual fear conditioning paradigm during the first mild shock whichtakes place at 4 min. Results are shown for mice that have undergoneearly-life stress versus normally reared control mice, and mice thathave undergone early-life stress and treated with the ellagitanninpunicalagin. Results are expressed as mean±SEM.

FIG. 43 is a bar graph depicting the extinction of a memory to aparticular adverse context when repeatedly exposed to the context in theabsence of the adverse effect. Data is shown for mice that haveundergone early-life stress, normally reared non-stressed control mice,and mice that have undergone early-life stress and treatment with theellagitannin punicalagin. Results are expressed as mean±SEM.Significance: *p<0.05, #p=0.05 (Student's t-test). Normal non-stressedanimals are compared to early-life stressed animals (i.e., maternalseparation). Punicalagin treated early-life stressed animals arecompared to untreated early-life stressed animals.

FIG. 44 is a line graph demonstrating the level of motor learning asmeasured by the latency in seconds to fall from a rotating rod. Data isshown for mice that have undergone early-life stress, normally rearedcontrol mice, and mice that have undergone early-life stress and havebeen treated with the ellagitannin punicalagin. Results are expressed asmean±SEM.

FIG. 45 is a graph depicting the escape latency in seconds from theMorris water maze during the training phase, a measurement of cognitivelearning. Data is shown for mice that have undergone early-life stress,normally reared control mice, and mice that have undergone early lifestress and have been treated with the ellagitannin punicalagin. Resultsare expressed as mean±SEM. Significance: * p<0.05 (Student's t-test).

FIG. 46 is a bar graph depicting the effects of pomegranate-derivedcompounds on contextual recognition in normal mice, either untreated ortreated with punicalagin or urolithin A. Results are expressed asmean±SEM. Significance: *p<0.05 (Student's t-test).

FIG. 47 is a bar graph depicting the effects of pomegranate-derivedcompounds on retention of memory for a particular context in normalmice, either untreated or treated with punicalagin or urolithin A.Results are expressed as mean±SEM. Significance: Data were analyzedusing either one-way ANOVA or repeated measure ANOVA, followed by aFisher post-hoc LSD multiple comparison test. *p<0.05.

FIG. 48 is a line graph demonstrating muscle performance and motorskills as measured by the latency to fall in seconds from a turningrotarod. Data is shown for normally reared untreated control mice andmice that have been treated with the ellagitannin punicalagin.Significance: * p<0.05 by ANOVA analysis.

FIG. 49 is a line graph demonstrating the level of muscle performanceand endurance as measured by ability of a mouse to run on a treadmill atelevated speeds. Data is shown for normally reared untreated controlmice and mice that have been treated with urolithin A. Significance:*p<0.05, **p<0.01 (Student's t-test).

DETAILED DESCRIPTION OF THE INVENTION

In biology and psychology, the term “stress” refers to the consequenceof the failure of a human or other animal to respond appropriately tophysiological, emotional, or physical threats, whether actual orimagined. The term “stress” was first employed in a biological contextby the endocrinologist Hans Selye in the 1930s. He later broadened andpopularized the concept to include inappropriate physiological responseto any demand. It covers a wide range of phenomena, from mild irritationto drastic dysfunction that may cause severe health breakdown.

All of these psychobiological features of stress may representmanifestations of oxidative stress, an imbalance between the productionand manifestation of reactive oxygen species and the ability of abiological system readily to detoxify the reactive intermediates or torepair the resulting damage. Disturbances in the normal redox state oftissues can cause toxic effects through the production of peroxides andfree radicals that damage all of the components of the cell, includingproteins, lipids, and DNA. Some reactive oxidative species can even actas messengers through a phenomenon called “redox signaling.”

In humans, oxidative stress is involved in many diseases. Examplesinclude atherosclerosis, Parkinson's disease, heart failure, myocardialinfarction, Alzheimer's disease, schizophrenia, bipolar disorder,fragile X syndrome, and chronic fatigue syndrome.

One source of reactive oxygen under normal conditions in humans is theleakage of activated oxygen from mitochondria during oxidativephosphorylation.

Other enzymes capable of producing superoxide (O₂ ⁻) are xanthineoxidase, NADPH oxidases and cytochromes P450. Hydrogen peroxide, anotherstrong oxidizing agent, is produced by a wide variety of enzymesincluding several oxidases. Reactive oxygen species play important rolesin cell signaling, a process termed redox signaling.

Thus, to maintain proper cellular homeostasis a balance must be struckbetween reactive oxygen production and consumption.

The best studied cellular antioxidants are the enzymes superoxidedismutase (SOD), catalase, and glutathione peroxidase. Less-well-studiedenzymatic antioxidants include the peroxiredoxins and the recentlydiscovered sulfiredoxin. Other enzymes that have antioxidant properties(although this role is not primary) include paraoxonase, glutathione-Stransferases, and aldehyde dehydrogenases.

Oxidative stress contributes to tissue injury following irradiation andhyperoxia. It is suspected to be important in neurodegenerativediseases, including Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis (ALS), and Huntington's disease. Oxidativestress is also thought to be linked to certain cardiovascular diseases,since oxidation of low-density lipoprotein (LDL) in the vascularendothelium is a precursor to plaque formation. Oxidative stress alsoplays a role in the ischemic cascade due to oxygen reperfusion injuryfollowing hypoxia. This cascade includes both strokes and heart attacks.Oxidative stress has also been implicated in chronic fatigue syndrome.

Remarkably, the inventors have discovered that certain compounds derivedfrom ellagitannins are useful in the treatment and prevention ofphysiological and psychological manifestations of stress, includingoxidative stress. Without meaning to be tied to any particular mechanismof action, it is believed that the compounds exert beneficial effects onmitochondria, promoting and restoring crucial mitochondrial functionsand counteracting stress-induced mitochondrial dysfunction. These samecompounds have been discovered, in accordance with the instantinvention, to be useful in the treatment and prevention of any of avariety of conditions, diseases, and disorders related to mitochondrialdysfunction including, without limitation, neurodegenerative diseasesand cognitive disorders, metabolic disorders including insulinresistance, mood disorders, and anxiety disorders.

Ellagitannins (ETs) are polyphenols included within the so called“hydrolyzable tannins” in which hexahydroxydiphenic acid forms diesterswith sugars (most often β-D-glucose). ETs can occur as complex polymersreaching molecular weights up to 4000 and higher. These polymers can behydrolyzed with acids or bases to yield ellagic acid (EA), which can beused indirectly to quantify ETs. EA in turn is a source of additionalmetabolic products including urolithins.

Many plant species containing ellagitannins have been used for thetreatment of diseases, particularly in Asia (Okuda et al., 2009). Theseinclude Agrimonia pilosa (agrimoniin), Camelia japonica (camelliatanninA), Cornus officinalis (cornussin A), Geranium thunbergii (geraniin),Geum japonicum (gemin-A), Liquidambar formosana (casuarictin), Mallotusjaponicus (mallotusinic acid), Oenothera erythrosepala (oenothein B),Punica granatum (pomegranate) (granatin B), Rosa rugosa (rugosin), andTerminalia chebula (chebulinic acid), among others. The main uses ofthese medicinal plants have been associated to their antioxidant,anti-diarrheic, anti-microbial, and immunomodulatory activities.

Ellagitannins are also present in significant amounts in many berries,including strawberries, red and black raspberries (Zafrilla et al.,2001), blueberries, and blackberries. Ellagitannins have also been foundin apples, cherries, cloudberries, cranberries, currants, grapes, lime,mango, pineapple, pomegranate, prune, rhubarbs. Serrano et al. (2009)Mol Nutr Food Res. 53:S310-29. The ellagitannin rubusuaviin C can beisolated from the leaves of the Chinese sweet tea Rubus suavissimus S.Lee. Ellagitannins have also been identified in appreciable amounts innuts, including walnuts (Fukuda et al., 2003), pistachios, cashew nuts,chestnuts, oak acorns (Cantos et al., 2003) pecans (Villarreal-Lozoya etal., 2007) and peanuts.

They are also abundant in pomegranates (Gil et al., 2000), and muscadinegrapes (Lee and Talcott, 2002) and are important constituents of wood,particularly oak (Glabasnia and Hofmann, 2006). Ellagitannins can beincorporated into food products, such as wines, and whiskey, throughmigration from wood to the food matrix during different aging processes.Ellagic acid has also been found in several types of honey and it hasbeen proposed as a floral marker for heather honey (Ferreres et al.,1996). Free ellagic acid and different glycosidic derivatives are alsopresent in these food products, including glucosides, rhamnosides,arabinosides and the corresponding acetyl esters (Zafrilla et al.,2001).

A number of studies have shown that the ellagitannin content of severalfood products can be quite high (Table 1). For example, a glass ofpomegranate juice (200 mL) can provide as much as 1 g of ellagitanninsand ellagic acid together, a raspberry serving (100 g raspberries)around 300 mg, a strawberry serving 70 mg, and four walnuts some 400 mgof ellagitannins.

Representative dietary ellagitannins include punicalagin of pomegranate,sanguiin-H-6 of strawberry and raspberry, and pedunculagin of walnuts.All of these release ellagic acid upon hydrolysis, although othermetabolites can also be produced and are distinctive of individualellagitannins (e.g., gallagic and ter-gallagic acids).

TABLE 1 Ellagitannins (ETs) and ellagic acid (EA) content in variousfood products. Food Content Fresh fruits Raspberry 263-330 mg/100 g f.w.Raspberry 51-330 mg/100 g f.w. Strawberry 77-85 mg/100 g f.w. Strawberry25 mg/100 g f.w. Cloudberry 315 mg/100 g f.w. Cloudberry 56-360 mg/100 gf.w. Blackberry 1.5-2.0 mg/g d.w. Arctic bramble 69-320 mg/100 g f.w.Pomegranates 35-75 mg/100 g f.w. (arils) Muscadine grapes 36-91 mg/100 gf.w. Nuts Walnut 802 mg/50 g (8 nuts) Pecan 20.96-86.2 mg/g (EA)Chestnut 1.61-24.9 mg/kg d.w. (EA) Processed fruits Pomegranate juice1500-1900 mg/L (punicalagin) Pomegranate juice 2020-2660 mg/L (ETs andEA) Pomegranate juice 5700 mg/L (ETs and EA) Raspberry jam 76 mg/100 gf.w. Strawberry jam 24 mg/100 g f.w. Muscadine grape juice 8-84 mg/LWines Oak-aged red wine 9.4 mg/L Oak-aged red wine 50 mg/L Muscadinegrape wine 2-65 mg/L Spirits Whiskey 1-2 mg/L Cognac 31-55 mg/L f.w.,fresh weight d.w., dry weight

Ellagitannins have an enormous structural variability, forming dimericand oligomeric derivatives. They also have a more widespreaddistribution than gallotanins. Additional ellagitannins and reportedsources for same are shown in Table 2.

TABLE 2 Other Ellagitannins. Molecular Weight Source Reference European(Puech, Mertz et Oak al. 1999) Heartwood 2-O-galloyl- punicalinCasaurictin Rhu tree, Wikipedia Stachyrus plant Castalagin & 934.63Pomegranate (Tanaka, Vecalagin bark Nonaka et al. 1986) CastalinCasuarictin T. japonica Casuariin Banaba tree (Bai, He et al. leaves2008) Casuarinin Banaba tree (Bai, He et al. leaves 2008) CasuarininPomegranate Chebulagic acid T. chebula Chebulinic acid T. chebulaCorilagin Pomegranate Cornusiin E Epipunicacortein Banaba tree (Bai, Heet al. A leaves 2008) Flosin B Banaba tree (Bai, He et al. leaves 2008)Gemin D T. japonica Granatin A Pomegranate Granatin B PomegranateGrandinin Lagerstroemin Banaba tree (Bai, He et al. leaves 2008)Lambertianin C Raspberries (Gasperotti, Masuero et al.) Pedunculagin784.52 Pomegrante (Tanaka, bark, and Nonaka pericarp et al. 1986)Punicacortein A Pomegranate Punicacortein B Pomegranate Punicacortein CPomegranate Punicacortein D Pomegranate Punicafolin PomegranatePunicalagin Pomegranate Punicalin Pomegranate Punigluconin PomegranateRoburin A Roburin B Roburin C Roburin D Roburin E Rubusuaviin C Tealeaves Sanguiin H-4 Muscadine (Lee, Johnson et grapes al. 2005) SanguiinH-5 Muscadine (Lee, Johnson et grapes al. 2005) Sanguiin H-6Raspberries, (Vrhovsek, Sanguisorba Palchetti et al. 2006) Sanguiin H-10Stachyurin Banaba tree (Bai, He et al. leaves 2008) StrictininTellimagrandin I Pomegranate Tellimigrandin II Pomegranate TerchebulinTerflavin A Terflavin B Tergallagin T. catappa Terminalin/ PomegranateGallagyldilacton

Many potentially active ellagitannins can be isolated from variousspecies of Terminalia plants. In particular, both punicalagin andpunicalin have been identified in several Terminalia species, including,e.g., T. catappa, T. chebula Retz, T. myriocarpa, and T. citrine.Punicalagin has also been isolated from Cistus salvifolius (aMediterranean shrub) and Combretum molle (an African shrub).

Ellagic acid is normally found in relatively low amounts in planttissues. Ellagic acid is thought to be derived from ellagitannins, whichwhen broken down form Hexahydroxydiphenic acid, which spontaneouslyconvert to ellagic acid. Some additional sources of ellagic acid areshown in Table 3.

TABLE 3 Sources with Ellagic Acid. Fruit Quantity Reference Acai 55.4 ±1.39 mg/L (Del Pozo-Insfran, fresh pulp Brenes et al. 2004) Umbu 314mg/100 g dry weight (De Souza Schmidt (commercial) Goncalves, Lajolo etal.) Camu-camu 490 mg/100 g dry weight (De Souza Schmidt Goncalves,Lajolo et al.) Cagaita 289 mg/100 g dry weight (De Souza Schmidt(commercial) Goncalves, Lajolo et al.) Araçá 262 mg/100 g dry weight (DeSouza Schmidt 218 mg/100 g dry weight Goncalves, Lajolo et al.)(commercial) Cambuci 240 mg/100 g dry weight (De Souza Schmidt 512mg/100 g dry weight Goncalves, Lajolo et al.) (commercial) Muscadine 219mg/100 g dry weight (Lee, Johnson Grapes et al. 2005)

Pomegranate (Punica granatum) fruits are ancient medicinal foods whichhave been used for centuries in folk medicine. They are consumed freshand as juice, which is an excellent source of ellagitannins and ellagicacid. Ellagitannins in pomegranate fruit husk and juice includepunicalin, punicalagin, corilagin, casuarinin,terminalin/gallagyldilacton, pedunculagin, tellimagrandin, granatin A,and granatin B. Other parts of the pomegranate plant contain additionalellagitannins, including punicafolin, punicacortein A, punicacortein B,punicacortein C, punicacortein D, and punigluconin. Commercial juicescontain gallagyl-type ellagitannins, including punicalagin isomers(1500-1900 mg/L), undefined hydrolyzable tannins (400-500 mg/L), andellagic acid and its glycosides (120-260 mg/L) (Gil et al., 2000).Punicalagins, ellagitannins in which gallagic and ellagic acids arelinked to a glucose molecule, are abundant in pomegranate peel.Punicalagin isomers and ellagic acid derivatives are not present in thearil juice, but during industrial juice processing they are extractedfrom the husk and membrane surrounding the arils and released in largequantities into the juice.

Extracts of the invention can be prepared by first juicing a fruit, forexample, the pomegranate may be juiced using standard industrial juicingmethods know in the art which may include juicing the whole fruit byapplication of pressure to the entire fruit or by first deshelling thepomegranate and then applying pressure to the remaining material,consisting of the arils, the membranous materials which entrap the arilsand the material of the husk produced during the deshelling process.Alternatively, the husk, which is a rich source of the ellagitannins, inparticular punicalagin, may undergo a juicing process that includes awater extraction. Alternative, non-water extraction methods may employother solvents such as ethanol, acetone, or methanol, as means ofexample.

The extract is typically an aqueous extract, which can consistessentially of the juice of the fruit, optionally with the addition ofextra water. Such aqueous extracts can be concentrated, enriched orcondensed by, for example, standard techniques, e.g., evaporation underreduced pressure and filtration methods. Examples of concentrates arethose which are at least 2-fold concentrated, more usually at least4-fold, for example at least 8-fold, at least 40-fold, at least100-fold, at least 200-fold, or at least 1000-fold.

The extracts can be fractionated to isolate one or more activecomponents therein by, for example, molecular weight filtration, orchromatography on a suitable solid support such as a sepharose gel (forsize exclusion chromatography) or ion-exchange column using HPLC on asuitably treated silica or alumina, for example ODS coated silica, or bysolvent extraction.

In vitro digestion simulation studies have shown that, in general,ellagitannins are quite stable under the physiological conditions of thestomach. The acidic conditions (HCl, pH 1.8-2.0) and the stomach enzymesdo not hydrolyze the original ellagitannins to release free ellagic acid(EA), and no degradation of the ellagitannins has been observed(Tomas-Barberan et al., 2009). While the stomach seems to be the firstimportant place for the absorption of free EA, ellagitannins are notabsorbed. Under the physiological conditions of the small intestine,however, there is a release of free EA from ellagitannins. Thishydrolysis seems to be due to the pH conditions (neutral to mildalkaline pH, 7.0-7.3) rather than to the effect of pancreatic enzymesand bile salts (Larrosa et al., 2006).

Animal studies have also been used to evaluate the bioavailability andmetabolism of EA and ellagitannins. A rapid absorption and metabolism ofEA was reported by Doyle and Griffiths (1980) in rats. These authorsdetected urolithin A (UA) and another metabolite (most probablyurolithin B (UB)) in feces and urine. Both UA and UB were demonstratedto be of microfloral origin since none were found in germ-free animals.Unchanged EA was not detected in urine or feces. These urolithins arelargely absorbed and glucuronidated by the intestinal cells. In thiscase, no methyl ethers are produced as UA and UB do not haveortho-dihydroxyl groupings in their molecules and therefore are notsubstrates for catechol-O-methyl transferase (COMT). In the case of UB,an additional hydroxyl can be introduced by cytochrome P450, and thisincreases the possibilities of glucuronidation and enhances theexcretion of the metabolite. Teel and Martin (1988) found that both freeEA and some conjugates (sulfate ester, glucuronide and glutathioneconjugates) were detected in mice urine, bile and blood. Absorption of³H-EA occurred mostly within two hours of oral administration. Levels inblood, bile and tissues were low, and absorbed compounds were excretedin urine. More than half of the administered ³H-EA remained in thegastrointestinal tract after 24 h.

The metabolism of various dietary ETs and EA derivatives has beenassessed in humans. In a study involving forty healthy volunteers,divided into four groups, different ET-containing foodstuffs wereadministered, including strawberries (250 g), red raspberries (225 g),walnuts (35 g), and oak-aged red wine (300 mL). Strawberries andraspberries both contain the ET sanguiin H-6; walnuts contain the ETpedunculagin; and oak-aged wine contains the ET vescalagin. After theintake, five urine fractions were collected at 8, 16, 32, 40, and 56 h.Neither ETs nor EA were detected in urine using LC-MS/MS analysis.However, the microbial metabolite3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin B) conjugated withglucuronic acid was detected among the fractions starting at 32 h until56 h in all of the subjects, independent of the consumed foodstuff.According to the results obtained, urolithin B derivatives were excretedindependently of the ET consumed. A common monomeric moiety in the ETsconsumed was EA (m/z- at 301), which could indicate that this subunitbelonging to ET molecules was the critical molecule to produce urolithinB derivatives. A similar metabolic transformation to ellagic acid andurolithin was observed for the ellagitannins in humans consumingpomegranate juice (Cerda, Espin et al. 2004; Cerda, Periago et al.2005).

One of the main factors in the metabolism and bioavailability ofellagitannins is their microbial transformation to render a series ofurolithin derivatives (FIG. 2). Among them, the best characterized andknown are urolithin A and B, but intermediates with three and fourhydroxyls are also produced in the small intestine, absorbed, andexcreted in the bile after conjugation with methyl ethers andglucuronides (Espin et al., 2007). Animal experiments show that thesemetabolites start to be formed in the small intestine, indicating thatanaerobic bacteria may be responsible for this. The metabolism continuesalong the GI tract starting with urolithins D and C to end with theproduction of urolithins A and B. Differences in the production of thesemetabolites by human volunteers show that they may be produced by theactivity of specific microorganisms present in the gut.

In the gastrointestinal tract and in other tissues (mainly in theliver), EA and ellagitannin microbial metabolites are furthermetabolized either by Phase I (hydroxylation) and Phase II (methylation,glucuronidation, and sulfation) enzymes to render more solublemetabolites that may be distributed among tissues and then excreted inurine.

Thus, UB can be hydroxylated to produce UA and this can be furtherhydroxylated to produce tri-hydroxy-derivatives.

Phase II products are also produced and methyl ethers (products of COMT)as well as different glucuronide conjugates are detected in differenttissues and in urine. Sulphate conjugates of ellagitannin metabolitesare less abundant in animals and humans than the glucuronide conjugates.These conjugates are first produced in the intestinal cells, and furthermetabolized in the liver before excretion in the urine or the bile.

To summarize, ellagitannins are generally not absorbed in the gut.Rather, they release EA in the gut, which is only poorly absorbed in thestomach and small intestine. EA is largely metabolized by unidentifiedbacteria in the intestinal lumen to produce urolithins. Microbialmetabolism starts in the small intestine and the first metabolitesproduced retain four phenolic hydroxyls (urolithin D, four hydroxylgroups), and these are further metabolized along the intestinal tract toremove hydroxyl units leading to urolithin C (three hydroxyls),urolithin A (two hydroxyls) and B (one hydroxyl) in the distal parts ofthe colon (FIG. 2). The absorbed metabolites are conjugated withglucuronic acid (one or two units), and/or methyl ethers (whenortho-dihydroxyl groupings are present). Urolithin A and B conjugatesare the main metabolites detected in plasma and urine, although sometrihydroxy derivatives (hydroxyl-UA) or EA-dimethyl ether glucuronidehave also been detected in smaller amounts. The tetrahydroxy-urolithins,trihydroxy-urolithins, and EA derivatives generally are not detected inperipheral plasma, but they are absorbed in the small intestine and theyare transported to the liver where they are further metabolized andexcreted with bile to the small intestine establishing an enterohepaticcirculation that is responsible for the relatively long life ofurolithins in plasma and urine.

In addition to natural food sources, many papers have appeared on thebiosynthesis, isolation, and biological activity of tannins, especiallyellagitannins, over the last twenty years (e.g., Xie et al., 1995,Yoshida et al., 1982, 1984, 1985, 1986, 1989, 1990a/b, 1991a-d, 1992a/b,1995, Nonaka et al., 1980, 1984, 1989a-c, 1990, Tanaka et al., 1986a/b,1990, 1992a/b, 2001, Hatano et al., 1988, 1989, 1990a-c, 1991, 1995, Linet al., 1990, Nishizawa et al., 1982, 1983, Haddock et al., 1982a/b,Kashiwada et al., 1992a/b, 1993, Kadota et al., 1990, Okuda et al.,1982a-e, 1983a/b, El-Mekkawy et al., Chemistry and Biology ofEllagitannins 154 1995, Tsai et al., 1992, Han et al., 1995, Chen etal., 1995, Morimoto et al., 1986a/b, Saijo et al., 1989). Access to pureellagitannins by isolation from natural sources may be cumbersome andyield only relatively small quantities of pure natural products. See,for example, Okuda et al., (1982) Chem Pharm Bull. 30:4230-4233; Okudaet al. (1982) Chem Pharm Bull. 30:234-4236. It is therefore notable thatmethods for total synthesis of many ellagitannins are known. See, forexample, Khanbabaee, K., Strategies for the synthesis of ellagitannins,In: Chemistry and Biology of Ellagitannins, Ed. S. Quideau, WorldScientific Publishing, Singapore, 2009, pp. 152-202, includingreferences cited therein.

Antioxidant activities of food extracts rich in ellagitannins have beendetermined by using various in vitro assays, and the high activities ofstrawberries (Meyers et al., 2003, Aaby et al., 2005, 2007), raspberries(Liu et al., 2002, Beekwilder et al., 2005), cloudberries (Kahkonen etal., 2001) and other Rubus berries (Wada and Ou, 2002), pomegranates(Gil et al., 2000) and walnuts (Anderson et al., 2001) and theirellagitannins have been extensively reported. These foods also rank highwhen compared to other plant-based foods.

Less is known about the effects of consumption of ellagitannin-richfoods on the antioxidant status in vivo. In elderly women, the totalantioxidant capacity of serum increased by about 10% during the 4-hourperiod after consumption of 240 g of strawberries (Cao et al., 1998). Asingle dose of standardized pomegranate extract (Mertens-Talcott et al.,2006) and long-term consumption of pomegranate juice (Rosenblat et al.,2006) also improved several antioxidant parameters in human volunteers.However, the daily consumption of walnuts for three weeks had no effecton the antioxidant status of subjects with metabolic syndrome (Davis etal., 2007).

Cancer cell growth is dependent on the balance between proliferation andapoptosis. Unregulated cell proliferation and suppression of apoptosisare key steps in initiation and progression of cancer. There is asubstantial amount of evidence that extracts of ellagitannin-rich foodsreduce the growth of cancer cells in vitro by inhibiting cellproliferation, inducing apoptotic cell death, and modulating cell cyclekinetics and signal transduction pathways.

In vitro studies carried out with cancer cell lines have shown thatstrawberries (Meyers et al., 2003, Olsson et al., 2004, Ramos et al.,2005, Wang et al., 2005, Wu et al., 2007), raspberries (Liu et al.,2002, Olsson et al., 2004, Wu et al., 2007), cloudberries (Wu et al.,2007) and rose hips (Olsson et al., 2004) inhibit cell proliferation,induce apoptosis and cause cell cycle arrest in human colon, liver,lung, breast or cervical cancer cells. In these studies, thecontribution of ellagitannins on the activities of berry extracts wasnot assessed. However, a recent study (Ross et al., 2007) suggests thatthe anti-proliferative activity of raspberries is predominantlyassociated with ellagitannins.

Pomegranate juice and its ellagitannins also have been reported toinhibit proliferation, induce apoptosis, and suppress inflammatory cellsignaling in colon cancer cell lines (Seeram et al., 2005, Adams et al.,2006, Larrosa et al., 2006). Likewise, polyphenols in muscadine grapeskin inhibit the growth of colon cancer cells and induce apoptosis (Yiet al., 2005). Fractions isolated from red muscadine grapes and rich inellagic acid, ellagic acid glycosides, and ellagitannins induceapoptosis, decrease cell number, and cause alterations in cell cyclekinetics in colon carcinoma cells (Mertens-Talcott et al., 2006).

Pomegranate fruit juice is effective against prostate cancer cells invitro, but not against normal prostate epithelial cells. Treatment ofhighly aggressive human prostate cancer cells with pomegranate fruitextract resulted in inhibition of cell growth and viability andinduction of apoptosis (Malik et al., 2005, Malik and Mukhtar, 2006).

In accordance with the instant invention, it has now been discoveredthat ellagitannins and their metabolites, including ellagic acid and,especially, urolithins, unexpectedly exhibit protective and restorativeeffects on mitochondria. Without meaning to be limited to any particularmechanism, it is believed that various types of stress result in stressinjury to mitochondria, thereby reducing their ability to performnumerous functions essential to overall cell function. The methods ofthe invention are useful for treating conditions involving stress injuryto mitochondria, which injury may be manifest in any of a number of waysincluding, but not limited to, mitochondrial disease.

Mitochondria are the “power centers” of cells. These double-membraneorganelles play a critical role in generating the vast majority ofcellular energy (ATP) via oxidative phosphorylation. Mitochondria arealso essential for other key metabolic functions, such as fatty acidβ-oxidation, catabolism of amino acids, ketogenesis, and generation ofreactive oxygen species (ROS) with important signaling functions andcontrol of calcium homeostasis.

The mitochondrial matrix contains the enzymatic machinery for fatty acid(3-oxidation, which generates acetyl-CoA from acyl chains, and reducingequivalents in the form of reduced nicotinamide adenine dinucleotide(NADH) and reduced flavin adenine dinucleotide (FADH2) in the process.Acetyl-CoA fuels the tricarboxylic acid (TCA) cycle, also known as thecitric acid cycle or Krebs cycle, which also produces NADH and FADH2.These products donate electrons to the electron transport chain (ETC),leading to the generation of a proton gradient across the innermitochondrial membrane. Dissipation of this gradient through themitochondrial ATP synthase generates energy in the form of ATP.

The ETC is composed of 4 large multisubunit complexes (complexes I toIV), which transport electrons generated by the TCA cycle to a finalacceptor, molecular oxygen (02), forming H₂O at complex IV. Thetransport of electrons is accompanied by release of large amounts offree energy, most of which is harnessed for the translocation of protons(H⁺) from the matrix to the intermembrane space (proton motive force);the remainder is dissipated as heat. The energy contained in the H⁺electrochemical gradient generated by the ETC is then coupled to ATPproduction as H⁺ flow back into the matrix through mitochondrial ATPsynthase. Thus, oxidative phosphorylation results from electrontransport, the generation of a proton gradient, and subsequent protonflux coupled to mitochondrial ATP synthase.

ROS can also activate uncoupling proteins (UCPs) that dissipate theproton gradient without producing ATP. UCPs are considered to be naturalregulators of this process, responding to and controlling ROS productionby mitigating the formation of a large proton gradient. Additionally,UCPs and respiration uncoupling are implicated in numerous importantphysiological and pathological processes, such as adaptivethermogenesis, regulation of fatty acid oxidation, participation ininflammation, prevention of ROS formation, glucose homeostasis, bodyweight regulation, and aging.

Citrate synthase is the initial enzyme of the mitochondrial TCA cycle.This enzyme catalyzes the reaction between acetyl coenzyme A (AcetylCoA) and oxaloacetic acid to form citric acid. The activity of thisenzyme reflects both mitochondrial biogenesis and mitochondrialoxidative phosphorylation, since its activity increases proportionallyto mitochondrial density (number of mitochondrial per cell) and theactivity of mitochondrial respiration. Consequently, citrate synthasemeasurement allows an overall assessment of mitochondrial functionalstatus, with higher activity indicating an increased oxidativephosphorylation and ATP synthesis and lower activity indicating thecontrary.

In order to better understand the underlying molecular mechanism leadingto the improvement of mitochondrial function, a profile of keymitochondrial genes (encoding mitochondrial and genomic DNA) coveringoxidative phosphorylation, mitochondrial chain complexes, TCA cycle,uncoupling proteins, transcriptional factors, co-factors andROS-scavenging proteins may be performed.

The conventional teaching in biology and medicine is that mitochondriafunction only as “energy factories” for the cell. However, more than 95%(2900 of 3000) of genes encoding mitochondrial proteins are involvedwith other functions tied to the specialized duties of thedifferentiated cells in which they reside. These duties evolve duringdevelopment from embryo to adult, and as tissues grow, mature, and adaptto the postnatal environment. These other, non-ATP-related functions areintimately involved with most of the major metabolic pathways used by acell to build, break down, and recycle its molecular building blocks.Cells cannot even make the RNA and DNA they need to grow and functionwith out mitochondria. The building blocks of RNA and DNA are purinesand pyrimidines. Mitochondria contain the rate-limiting enzymes forpyrimidine biosynthesis (dihydroorotate dehydrogenase) and hemesynthesis (d-amino levulinic acid synthetase) required to makehemoglobin. In the liver, mitochondria are specialized to detoxifyammonia in the urea cycle. Mitochondria are also required forcholesterol metabolism, for estrogen and testosterone synthesis, forneurotransmitter metabolism, and for free radical production anddetoxification. Mitochondria do all this in addition to oxidizing thefat, protein, and carbohydrates ingested in the diet.

Mitochondrial diseases are the result of either inherited or spontaneousmutations in mitochondrial DNA or nuclear DNA which lead to alteredfunctions of the proteins or RNA molecules that normally reside inmitochondria. Problems with mitochondrial function, however, may onlyaffect certain tissues as a result of factors occurring duringdevelopment and growth that are not yet fully understood. Even whentissue-specific isoforms of mitochondrial proteins are considered, it isdifficult to explain the variable patterns of affected organ systems inthe mitochondrial disease syndromes seen clinically.

Mitochondrial diseases result from failures of the mitochondria,specialized compartments present in every cell of the body except redblood cells. Mitochondria are responsible for creating more than 90% ofthe energy needed by the body to sustain life and support growth. Whenthey fail, less and less energy is generated within the cell. Cellinjury and even cell death follow. If this process is repeatedthroughout the body, whole systems begin to fail, and the life of theperson in whom this is happening is severely compromised. Mitochondrialdiseases primarily affect children, but adult onset is becoming morerecognized.

Diseases of the mitochondria appear to cause the most damage to cells ofthe brain, heart, liver, skeletal muscles, kidney, and the endocrine andrespiratory systems.

Many symptoms in mitochondrial disorders are non-specific. The symptomsmay also show an episodic course, with periodic exacerbations. Theepisodic condition of migraine, as well as myalgia, gastrointestinalsymptoms, tinnitus, depression, chronic fatigue, and diabetes, have beenmentioned among the various manifestations of mitochondrial disorders inreview papers on mitochondrial medicine (Chinnery and Turnbull (1997)QJM 90:657-67; Finsterer (2004) Eur J Neurol. 11:163-86). In patientswith mitochondrial disorders, clinical symptomatology typically occursat times of higher energy demand associated with physiologicalstressors, such as illness, fasting, over-exercise, and environmentaltemperature extremes. Furthermore, psychological stressors alsofrequently trigger symptomatology, presumably due to higher brain energydemands for which the patient is unable to match with sufficient ATPproduction.

Depending on which cells are affected, symptoms may include loss ofmotor control, muscle weakness and pain, gastro-intestinal disorders andswallowing difficulties, poor growth, cardiac disease, liver disease,diabetes, respiratory complications, seizures, visual/hearing problems,lactic acidosis, developmental delays and susceptibility to infection.

Mitochondrial diseases include, without limitation, Alper's disease;Barth syndrome; beta-oxidation defects; carnitine deficiency;carnitine-acyl-carnitine deficiency; chronic progressive externalophthalmoplegia syndrome; co-enzyme Q10 deficiency; Complex Ideficiency; Complex II deficiency; Complex III deficiency; Complex IVdeficiency; Complex V deficiency; CPT I deficiency; CPT II deficiency;creatine deficiency syndrome; cytochrome c oxidase deficiency; glutaricaciduria type II; Kearns-Sayre syndrome; lactic acidosis; LCHAD(long-chain acyl-CoA dehydrogenase deficiency); Leber's hereditary opticneuropathy; Leigh disease; lethal infantile cardiomyopathy; Luftdisease; MAD (medium-chain acyl-CoA dehydrogenase deficiency);mitochondrial cytopathy; mitochondrial DNA depletion; mitochondrialencephalomyopathy, lactic acidosis, and stroke-like symptoms;mitochondrial encephalopathy; mitochondrial myopathy; mitochondrialrecessive ataxia syndrome; muscular dystrophies, myoclonic epilepsy andragged-red fiber disease; myoneurogenic gastrointestinal encephalopathy;neuropathy, ataxia, retinitis pigmentosa, and ptosis; Pearson syndrome;POLG mutations; pyruvate carboxylase deficiency; pyruvate dehydrogenasedeficiency; SCHAD (short-chain acyl-CoA dehydrogenase deficiency); andvery long-chain acyl-CoA dehydrogenase deficiency.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of pomegranate extract: for the treatmentor prevention of a condition selected from the group consisting ofobesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

As used herein, a “food product” refers to a product prepared from anatural food. Non-limiting examples of food products include juices,wines, concentrates, jams, jellies, preserves, pastes, and extracts. Asused herein, a “nutritional supplement” refers to a product suitable forconsumption or other administration principally for its health-promotingproperties rather than its caloric content.

As used herein, the term “metabolic syndrome” refers to a combination ofmedical disorders that, when occurring together, increase the risk ofdeveloping cardiovascular disease and diabetes. It affects one in fivepeople in the United States and prevalence increases with age. Somestudies have shown the prevalence in the United States to be anestimated 25% of the population. In accordance with the InternationalDiabetes Foundation consensus worldwide definition (2006), metabolicsyndrome is central obesity plus any two of the following:

-   -   Raised triglycerides: >150 mg/dL (1.7 mmol/L), or specific        treatment for this lipid abnormality;    -   Reduced HDL cholesterol: <40 mg/dL (1.03 mmol/L) in males, <50        mg/dL (1.29 mmol/L) in females, or specific treatment for this        lipid abnormality;    -   Raised blood pressure: systolic BP>130 or diastolic BP>85 mm Hg,        or treatment of previously diagnosed hypertension; and    -   Raised fasting plasma glucose: (FPG)>100 mg/dL (5.6 mmol/L), or        previously diagnosed type 2 diabetes.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of an ellagitannin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

In certain embodiments in accordance with this and other aspects of theinvention, the ellagitannin is selected from the group consisting of2-O-galloyl-punicalin, casaurictin, castalagin & vecalagin, castalin,casuarictin, casuariin, casuarinin, chebulagic acid, chebulinic acid,corilagin, cornusiin E, epipunicacortein A, flosin B, gemin D, granatinA, granatin B, grandinin, lagerstroemin, lambertianin C, pedunculagin,punicacortein A, punicacortein B, punicacortein C, punicacortein C,punicacortein D, punicafolin, punicalagin, punicalin, punigluconin,roburin A, roburin B, roburin C, roburin D, roburin E, rubusuaviin C,sanguiin H-4, sanguiin H-5, sanguiin H-6, sanguiin H-10, stachyurin,strictinin, tellimagrandin I, tellimigrandin II, terchebulin, terflavinA, terflavin B, Tergallagin, and terminalin/gallagyldilacton. Of course,additional ellagitannins are also contemplated by the invention.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of punicalagin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of ellagic acid: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

An aspect of the invention is a food product or nutritional supplementcomprising an effective amount of a urolithin: for the treatment orprevention of a condition selected from the group consisting of obesity,reduced metabolic rate, metabolic syndrome, diabetes mellitus,cardiovascular disease, hyperlipidemia, neurodegenerative disease,cognitive disorder, mood disorder, stress, and anxiety disorder; forweight management; or to increase muscle performance or mentalperformance.

In certain embodiments in accordance with this and other aspects of theinvention, the urolithin is urolithin A. In certain embodiments inaccordance with this and other aspects of the invention, the urolithinis urolithin B. In certain embodiments in accordance with this and otheraspects of the invention, the urolithin is urolithin C. In certainembodiments in accordance with this and other aspects of the invention,the urolithin is urolithin D.

In each of the foregoing, in one embodiment the condition is obesity.

In each of the foregoing, in one embodiment the condition is reducedmetabolic rate.

In each of the foregoing, in one embodiment the condition is metabolicsyndrome.

In each of the foregoing, in one embodiment the condition is diabetesmellitus.

In each of the foregoing, in one embodiment the condition iscardiovascular disease.

In each of the foregoing, in one embodiment the condition ishyperlipidemia.

In each of the foregoing, in one embodiment the condition isneurodegenerative disease.

In each of the foregoing, in one embodiment the condition is cognitivedisorder.

In each of the foregoing, in one embodiment the condition is mooddisorder.

In each of the foregoing, in one embodiment the condition is stress.

In each of the foregoing, in one embodiment the condition is anxietydisorder.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for weight management.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for increasing muscle performance.

In each of the foregoing, in one embodiment the food product ornutritional supplement is for increasing mental performance.

An aspect of the invention is a method of increasing or maintainingmitochondrial function. The method includes the step of contacting cellswith an effective amount of a urolithin or a precursor thereof, toincrease function of the mitochondria.

An aspect of the invention is a method of treating, preventing, ormanaging a mitochondria-related disease or condition associated with analtered mitochondrial function or a reduced mitochondrial density. Themethod includes the step of administering to a subject in need thereof atherapeutically effective amount of a urolithin or a precursor thereof,to treat the disease or condition associated with altered mitochondrialfunction or reduced mitochondrial density.

An aspect of the invention is a method of increasing metabolic rate. Themethod includes the step of administering to a subject in need thereofan effective amount of a urolithin or a precursor thereof, to increasemetabolic rate. As described elsewhere herein, precursors of a urolithincan include, without limitation, an ellagitannin, punicalagin, andellagic acid.

An aspect of the invention is a method of preventing or treatingmetabolic syndrome. The method includes the step of administering to asubject in need thereof an effective amount of a urolithin or aprecursor thereof, to prevent or treat metabolic syndrome.

An aspect of the invention is a method of preventing or treatingobesity. The method includes the step of administering to a subject inneed thereof an effective amount of a urolithin or a precursor thereof,to prevent or treat obesity.

An aspect of the invention is a method of preventing or treatingcardiovascular disease. The method includes the step of administering toa subject in need thereof an effective amount of a urolithin or aprecursor thereof, to prevent or treat cardiovascular disease.

An aspect of the invention is a method of treating hyperlipidemia. Themethod includes the step of administering to a subject in need thereofan effective amount of a urolithin or a precursor thereof, to treathyperlipidemia. In one embodiment, the hyperlipidemia ishypertriglyceridemia. In one embodiment, the hyperlipidemia is elevatedfree fatty acids.

An aspect of the invention is a method of treating a metabolic disorder.The method includes the step of administering to a subject in needthereof a therapeutically effective amount of a urolithin or a precursorthereof, to treat the metabolic disorder. In one embodiment, themetabolic disorder is diabetes mellitus. In one embodiment, themetabolic disorder is obesity.

Aging

By far the greatest risk factor for neurodegenerative diseases, such asAlzheimer's disease (AD), Parkinson's disease (PD), and amyotrophiclateral sclerosis (ALS), is aging. Mitochondria have been thought tocontribute to aging through the accumulation of mitochondrial DNA(mtDNA) mutations and net production of reactive oxygen species (ROS).Although most mitochondrial proteins are encoded by the nuclear genome,mitochondria contain many copies of their own DNA. Human mtDNA is acircular molecule of 16,569 base pairs that encodes 13 polypeptidecomponents of the respiratory chain, as well as the rRNAs and tRNAsnecessary to support intramitochondrial protein synthesis using its owngenetic code. Inherited mutations in mtDNA are known to cause a varietyof diseases, most of which affect the brain and muscles—tissues withhigh energy requirements. It has been hypothesized that somatic mtDNAmutations acquired during aging contribute to the physiological declinethat occurs with aging and aging-related neurodegeneration. It is wellestablished that mtDNA accumulates mutations with aging, especiallylarge-scale deletions and point mutations. In the mtDNA control region,point mutations at specific sites can accumulate to high levels incertain tissues: T414G in cultured fibroblasts, A189G and T408A inmuscle, and C150T in white blood cells. However, these control-region“hot spots” have not been observed in the brain. Point mutations atindividual nucleotides seem to occur at low levels in the brain,although the overall level may be high. Using a polymerase chainreaction (PCR)-cloning-sequencing strategy, it was found that theaverage level of point mutations in two protein-coding regions of brainmtDNA from elderly subjects was ˜2 mutations per 10 kb. Noncodingregions, which may be under less selection pressure, potentiallyaccumulate between twice and four times as many. The accumulation ofthese deletions and point mutations with aging correlates with declinein mitochondrial function. For example, a negative correlation has beenfound between brain cytochrome oxidase activity and increasedpoint-mutation levels in a cytochrome oxidase gene (CO1).

Net production of ROS is another important mechanism by whichmitochondria are thought to contribute to aging. Mitochondria containmultiple electron carriers capable of producing ROS, as well as anextensive network of antioxidant defenses. Mitochondrial insults,including oxidative damage itself, can cause an imbalance between ROSproduction and removal, resulting in net ROS production. The importanceto aging of net mitochondrial ROS production is supported byobservations that enhancing mitochondrial antioxidant defenses canincrease longevity. In Drosophila, overexpression of the mitochondrialantioxidant enzymes manganese superoxide dismutase (MnSOD) andmethionine sulfoxide reductase prolongs lifespan. This strategy is mostsuccessful in short-lived strains of Drosophila, and has no effect inalready long-lived strains. However, it has recently been shown thatoverexpression of catalase experimentally targeted to mitochondriaincreased lifespan in an already long-lived mouse strain.

Cognitive decline during aging has been observed to occur in aginganimals and is thought to occur as a result of changes in the synapticphysiology of aging neurons. These changes are thought to lead to anoverall global loss of integrative function of the neuronal signaling inthe brain (Bishop, Lu et al. 2010) and increased susceptibility to thelong-term effects of oxidative stress and inflammation (Joseph,Shukitt-Hale et al. 2005). Cell loss which takes place during normalaging is thought to occur primarily due to oxidative stress as a resultof free radicals produced by an inefficient and partially uncoupledoxidative pathway. Indeed, it has been shown that a common trait inaging among different species, (C. elegans, D. melanogaster, mice, rats,chimpanzees, and humans) has been evidence of reduced mitochondrialfunction. This interpretation is further validated by the observationthat significant impairment of mitochondrial function shortens lifespanin both C. elegans (Rea, Ventura et al. 2007) and mice (Trifunovic,Wredenberg et al. 2004; Kujoth, Hiona et al. 2005). Improvement ofmitochondrial function, through the overexpression of catalase in mice,resulted in extended life spans (Schriner, Linford et al. 2005).

With aging and the decline of mitochondrial function, neurons in thebrain become more vulnerable to age-dependent pathologies, as well ascell death. This results in a loss of connections between neurons, aswell as impaired neuronal function (loss of neurotransmitters, absenceof firing). There is also increased evidence that neurons respond tounrepaired DNA damage by silencing gene expression through epigeneticmechanisms, thus leading to further suppression of cell functions.Additionally, aging neuronal cells show in all species an increasedexpression of genes involved in stress response pathways.

Many hallmarks of these changes are observed in in vitro culture ofaging neuronal cells, which show decreased neurite outgrowth and processformation. A decrease that could be reversed by neuronal growth factors(Rozovsky, Wei et al. 2005).

Neurodegenerative Disorders

Neurodegenerative diseases are a heterogeneous group of disorderscharacterized by gradually progressive, selective loss of anatomicallyor physiologically related neuronal systems. Prototypical examplesinclude Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophiclateral sclerosis (ALS), and Huntington's disease (HD).

The early stages of neurodegeneration share many of the same hallmarksas the decline seen in aging. It is interesting to note that diseasessuch as Alzheimer's disease show an increased incidence with age, withover 50% of adults over the age of 85 presenting with the disease(Hebert, Scherr et al. 2003). As discussed above, decliningmitochondrial function appears to be a hallmark of aging. This declinein neuronal function is likely to have a significant impact on neuronalpopulations with large bioenergetic demands, one such set of neurons arethe large pyramidal neurons which degenerate in Alzheimer's disease(Bishop, Lu et al. 2010). The decline of these classes of neurons inresponse to impaired mitochondrial function may be responsible for theonset of neurodegenerative disease. The effects of neurodegenerativedisorders on neuronal survival can be modeled in vitro. When N2 neuronalcells are incubated with A-beta (Aβ) peptide, which is thought to be thecausative agent of Alzheimer's disease, there is a significant impact onneurite outgrowth, which can be reversed by anti-oxidants. Manczak etal. (2010) J Alzheimers Dis. 20 Suppl 2:S609-31.

The most common form of cell death in neurodegeneration is via theintrinsic mitochondrial apoptotic pathway. This pathway controls theactivation of caspase-9 by regulating the release of cytochrome c fromthe mitochondrial intermembrane space. The concentration of ROS, normalbyproducts of mitochondrial respiratory chain activity, is mediated inpart by mitochondrial antioxidants, such as manganese superoxidedismutase (SOD2) and glutathione peroxidase. Overproduction of ROS(oxidative stress) is a central feature of all neurodegenerativedisorders. In addition to the generation of ROS, mitochondria are alsoinvolved with life-sustaining functions, including calcium homeostasis,mitochondrial fission and fusion, the lipid concentration of themitochondrial membranes, and the mitochondrial permeability transition(MPT). Mitochondrial disease leading to neurodegeneration is likely, atleast on some level, to involve all of these functions (DiMauro andSchon, 2008).

There is evidence that mitochondrial dysfunction and oxidative stressplay a causal role in neurodegenerative disease pathogenesis, includingin four of the more well known diseases: Alzheimer's disease,Parkinson's disease, Huntington's disease, and amyotrophic lateralsclerosis (also known as Lou Gehrig's disease).

Alzheimer's disease (AD) is characterized clinically by progressivecognitive decline, and pathologically by the presence of senile plaquescomposed primarily of amyloid-β peptide (Aβ) and neurofibrillary tanglesmade up mainly of hyperphosphorylated tau. About 5-10% of cases arefamilial, occurring in an early-onset, autosomal-dominant manner. Threeproteins are known to be associated with such familial cases: amyloidprecursor protein (APP)—which is cleaved sequentially by β- andγ-secretases to produce Aβ—and presenilins 1 and 2 (PS1 and PS2), one orthe other of which is a component of each γ-secretase complex. Extensiveliterature supports a role for mitochondrial dysfunction and oxidativedamage in the pathogenesis of AD. Oxidative damage occurs early in theAD brain, before the onset of significant plaque pathology. Oxidativedamage also precedes Aβ deposition in transgenic APP mice, withupregulation of genes relating to mitochondrial metabolism and apoptosisoccurring even earlier and co-localizing with the neurons undergoingoxidative damage.

Several pathways connecting oxidative stress and AD pathology haverecently been uncovered. Oxidative stress may activate signalingpathways that alter APP or tau processing. For example, oxidative stressincreases the expression of β-secretase through activation of c-Junamino-terminal kinase and p38 mitogen-activated protein kinase (MAPK),and increases aberrant tau phosphorylation by activation of glycogensynthase kinase 3. Oxidant-induced inactivation of critical moleculesmay also be important. In a proteomic study, the prolyl isomerase PIN1was found to be particularly sensitive to oxidative damage. PIN1catalyses protein conformational changes that affect both APP and tauprocessing. Knockout of Pin1 increases amyloidogenic APP processing andintracellular AP levels in mice. Pin1-knockout mice also exhibit tauhyperphosphorylation, motor and behavioral deficits, and neuronaldegeneration. Oxidative induced damage of PIN1 and similarly sensitiveproteins could thus be important in promoting neurodegenerativeprocesses.

Mitochondria also play an important role in Parkinson's disease (PD)which is characterized clinically by progressive rigidity, bradykinesiaand tremor, and pathologically by loss of pigmented neurons in thesubstantia nigra and the presence of Lewy bodies—distinctive cytoplasmicinclusions that immunostain for α-synuclein and ubiquitin.

Mitochondria were first implicated in PD because MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine), whose metabolite MPP+ inhibitscomplex I of the mitochondrial electron-transport chain, causedparkinsonism in designer-drug abusers. This model has since been refinedin laboratory animals, in which chronic infusion of rotenone—anothercomplex-I inhibitor—or MPTP results clinically in a parkinsonianphenotype and pathologically in nigral degeneration with cytoplasmicinclusions immunoreactive for α-synuclein and ubiquitin. The mechanismof toxicity in these complex-I inhibition models probably involvesoxidative stress. Complex-I inhibition and oxidative stress were shownto be relevant to naturally occurring PD when complex-I deficiency andglutathione depletion were found in the substantia nigra of patientswith idiopathic PD and in patients with pre-symptomatic PD.

Many of the genes associated with PD also implicate mitochondria indisease pathogenesis. So far, mutations or polymorphisms in mtDNA and atleast nine named nuclear genes have been identified as causing PD oraffecting PD risk: α-synuclein, parkin, ubiquitin carboxy-terminalhydrolase L1, DJ-1, phosphatase and tensin homologue (PTEN)-inducedkinase 1 (PINK1), leucine-rich-repeat kinase 2 (LRRK2), the nuclearreceptor NURR1, HTRA2, and tau. Of the nuclear genes, α-synuclein,parkin, DJ-1, PINK1, LRRK2, and HTRA2 directly or indirectly involvemitochondria. In a small number of cases, inherited mtDNA mutationsresult in parkinsonism, typically as one feature of a larger syndrome.In one family, the Leber's optic atrophy G11778A mutation was associatedwith 1-DOPA-responsive parkinsonism, variably co-occurring withdementia, dystonia, ophthalmoplegia and ataxia. Notably, this mutationis in a subunit of complex I. Mutations in the nuclear-encoded mtDNApolymerase-γ (POLG) gene impair mtDNA replication and result in multiplemtDNA deletions, typically causing chronic progressive externalophthalmoplegia and myopathy. In such families, POLG mutations alsocosegregate with parkinsonism.

Amyotrophic lateral sclerosis (ALS) is characterized clinically byprogressive weakness, atrophy and spasticity of muscle tissue,reflecting the degeneration of upper and lower motor neurons in thecortex, brainstem and spinal cord. Approximately 90% of cases aresporadic (SALS) and 10% are familial (FALS). About 20% of familial casesare caused by mutations in Cu/Zn-superoxide dismutase (SOD1). In bothSALS and FALS, postmortem and biopsy samples from the spinal cord,nerves and muscles show abnormalities in mitochondrial structure, numberand localization. Defects in activities of respiratory chain complexeshave also been detected in muscle and spinal cord.

Huntington's disease (HD) is characterized clinically by chorea,psychiatric disturbances, and dementia, and pathologically by loss oflong projection neurons in the cortex and striatum. HD is inherited inan autosomal dominant manner, and is due to expansion of a CAGtrinucleotide repeat in the huntingtin (HTT) gene, which gives rise toan expanded polyglutamine stretch in the corresponding protein. Thenormal number of CAG (Q) repeats is less than 36; repeat numbers greaterthan 40 are associated with human disease. Various lines of evidencedemonstrate the involvement of mitochondrial dysfunction in HD. Nuclearmagnetic resonance spectroscopy reveals increased lactate in the cortexand basal ganglia. Biochemical studies show decreased activities ofcomplexes II and III of the electron-transport chain in the human HDbrain. In striatal cells from mutant Hu-knock-in mouse embryos,mitochondrial respiration and ATP production are significantly impaired.

An aspect of the invention is a method of treating a neurodegenerativedisease, age-related neuronal death or dysfunction. As used herein,“neurodegenerative disease” or, equivalently, “neurodegenerativedisorder”, refers to any condition involving progressive loss offunctional neurons in the central nervous system. In one embodiment, theneurodegenerative disease is associated with age-related cell death.Exemplary neurodegenerative diseases include, without limitation,Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (also known as ALS and as Lou Gehrig'sdisease), as well as AIDS dementia complex, adrenoleukodystrophy,Alexander disease, Alper's disease, ataxia telangiectasia, Battendisease, bovine spongiform encephalopathy (BSE), Canavan disease,corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewybodies, fatal familial insomnia, frontotemporal lobar degeneration,Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease,multiple sclerosis, multiple system atrophy, neuroacanthocytosis,Niemann-Pick disease, Pick's disease, primary lateral sclerosis,progressive supranuclear palsy, Refsum disease, Sandhoff disease,diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacutecombined degeneration of spinal cord, tabes dorsalis, Tay-Sachs disease,toxic encephalopathy, transmissible spongiform encephalopathy, andwobbly hedgehog syndrome.

In one embodiment, the method is used to treat age-related neuronaldeath or dysfunction. Such method is directed to neurodegeneration thatis not attributable to a specific neurodegenerative disease, e.g.,Alzheimer's disease, amyotrophic lateral sclerosis, Huntington'sdisease, and Parkinson's disease.

In one embodiment, a neurodegenerative disease is selected from thegroup consisting of Alzheimer's disease, amyotrophic lateral sclerosis,Huntington's disease, and Parkinson's disease.

In one embodiment, a neurodegenerative disease is Alzheimer's disease.

The method includes the step of administering to a subject in need oftreatment of a neurodegenerative disease a therapeutically effectiveamount of a urolithin or a precursor thereof, thereby treating theneurodegenerative disease.

In accordance with this and other methods of the invention, a“urolithin,” as used herein, refers to any one or combination ofurolithin A, urolithin B, urolithin C, and urolithin D (see, forexample, FIG. 1 and FIG. 2). In one embodiment, a urolithin is urolithinA, urolithin B, urolithin C, urolithin D, or any combination ofurolithin A, urolithin B, urolithin C, and urolithin D. In oneembodiment, a urolithin is urolithin A, urolithin B, or a combination ofurolithin A and urolithin B. In one embodiment, a urolithin is urolithinA. In one embodiment, a urolithin is provided as an isolated urolithin,e.g., isolated from a natural source or prepared by total synthesis.Isolated urolithins may be synthesized de novo. See, for example, USPat. Application Publication No. 2008/0031862 to Ghosal, the entirecontents of which are incorporated herein by reference.

In one embodiment, urolithin A (3,8-Dihydroxydibenzo-α-pyrone) wassynthesized in a two-stage synthesis as follows. Stage 1 is acopper-catalyzed reaction that occurs in the presence of a base (Hurtleyreaction) where the starting materials 2-bromo-5-methoxybenzoic acid andresoricinol are reacted together to generate the dihydro-dibenzopyronescaffold. In Stage 2 the demethylation of the benzopyrone with BBr₃yields 3,8-Dihydroxydibenzo-α-pyrone (urolithin A).

A mixture of 2-bromo-5-methoxybenzoic acid 1 (27.6 g), resorcinol 2(26.3 g) and sodium hydroxide (10.5 g) in water (120 mL) was heatedunder reflux for 1 hour. A 5% aqueous solution of copper sulfate (3.88 gof CuSO₄, 5H₂O in 50 mL water) was then added and the mixture wasrefluxed for additional 30 minutes. The mixture was allowed to cool downto room temperature and the solid was filtered on a Büchner filter. Theresidue was washed with cold water (50 mL) to give a pale red solid(38.0 g) which was triturated in hot MeOH (200 mL). The suspension wasleft overnight at 4° C. The resultant light red precipitate was filteredand washed with cold MeOH (75 mL) to yield the title compound 3 as apale brown solid. ¹H NMR is in accordance with the structure of 3.

To a suspension of 3 (10.0 g; 41 mmol; 1.0 eq.) in dry dichloromethane(100 mL) was added at 0° C. a 1 M solution of boron tribromide in drydichloromethane (11.93 mL of pure BBr₃ in 110 mL of anhydrousdichloromethane). The mixture was left at 0° C. for 1 hour and was thenallowed to warm up to room temperature. The solution was stirred at thattemperature for 17 hours. The yellow precipitate was filtered and washedwith cold water (50 mL) to give a yellow solid which was heated toreflux in acetic acid (400 mL) for 3 hours. The hot solution wasfiltered quickly and the precipitate was washed with acetic acid (50 mL)then with diethyl ether (100 mL) to yield the title compound 4 as ayellow solid. Structure and purity were determined by ¹H and ¹³C-NMR.

In one embodiment, a “urolithin” as used herein is or can include aglucuronated, methylated, or sulfated urolithin.

In accordance with this and other methods of the invention, a “urolithinprecursor,” as used herein, refers to an ellagitannin or an ellagitanninmetabolite, including but not limited to ellagic acid (EA). In oneembodiment, a urolithin precursor is punicalagin (PA). In oneembodiment, a urolithin precursor is punicalin (PB). See, for example,FIG. 1. In one embodiment, a urolithin precursor is ellagic acid (EA).In one embodiment, a urolithin precursor is provided as an isolatedurolithin precursor, e.g., isolated from a natural food source orprepared by total synthesis. Isolated urolithin precursors are usuallypurified from natural sources or synthesized de novo; some urolithinprecursors, including EA, are commercially available from suppliers,such as Sigma Aldrich.

Also in accordance with this and other methods of the invention,precursors of urolithins also include natural foods containingellagitannins and ellagic acid, especially natural foods that are richin ellagitannins, ellagic acid, or both ellagitannins and ellagic acid.Such foods include certain berries, grapes, pomegranates, rose hips, andnuts. In one embodiment, the natural food is pomegranate.

Additionally, precursors of urolithins include processed foods anddrinks prepared from such natural foods. The processed food can take anyform, including, for example, jams, jellies, preserves, pastes, spreads,juices, wines, extracts, concentrates, and the like.

In one embodiment, the processed food is pomegranate juice.

In one embodiment, a urolithin precursor is provided as an extract,e.g., a fruit extract.

In one embodiment, a urolithin precursor is provided as concentrate,e.g., a fruit concentrate or fruit juice concentrate.

The method of the invention can be used alone or in combination with anymethod or compound known to be useful to treat neurodegenerativedisease. For example, In one embodiment, the method of the invention canbe combined with use of any one or more of acetylcholinesteraseinhibitors, such as donezepil (Aricepe), galantamine (Razadyne), andrivastigmine (Exelon®), and N-methyl D-aspartate (NMDA) receptorantagonists, such as memantine (Namende).

An aspect of the invention is a method of improving cognitive function.As used herein, “cognitive function” refers to any mental process thatinvolves symbolic operations, e.g., perception, memory, attention,speech comprehension, speech generation, reading comprehension, creationof imagery, learning, and reasoning. In one embodiment, “cognitivefunction” refers to any one or more of perception, memory, attention,and reasoning. In one embodiment, “cognitive function” refers to memory.

The method includes the step of administering to a subject in need ofimproved cognition a therapeutically effective amount of a urolithin ora precursor thereof, thereby improving cognitive function.

Methods for measuring cognitive function are well known and can include,for example, individual or battery tests for any aspect of cognitivefunction. One such test is the Prudhoe Cognitive Function Test.Margallo-Lana et al. (2003) J Intellect Disability Res. 47:488-492.Another such test is the Mini Mental State Exam (MMSE), which isdesigned to assess orientation to time and place, registration,attention and calculation, recall, language use and comprehension,repetition, and complex commands. Folstein et al. (1975) J Psych Res.12:189-198. Other tests useful for measuring cognitive function includethe Alzheimer Disease Assessment Scale-Cognitive (ADAS-Cog) (Rosen etal. (1984) Am J Psychiatry. 141(11):1356-64) and the CambridgeNeuropsychological Test Automated Battery (CANTAB) (Robbins et al.(1994) Dementia. 5(5):266-81). Such tests can be used to assesscognitive function in an objective manner, so that changes in cognitivefunction, for example in response to treatment in accordance withmethods of the invention, can be measured and compared.

The method of the invention can be used alone or in combination with anymethod or compound known to improve cognitive function. For example, Inone embodiment, the method of the invention is combined with use ofcaffeine or nicotine or both.

In one embodiment, the subject does not have a cognitive disorder. Forexample, the method can be used to enhance cognitive function in asubject having normal cognitive function.

An aspect of the invention is a method of treating a cognitive disorder.As used herein, a cognitive disorder refers to any condition thatimpairs cognitive function. In one embodiment, “cognitive disorder”refers to any one or more of delirium, dementia, learning disorder,attention deficit disorder (ADD), and attention deficit hyperactivitydisorder (ADHD). In one embodiment, the cognitive disorder is a learningdisorder. In one embodiment, the cognitive disorder is attention deficitdisorder (ADD). In one embodiment, the cognitive disorder is attentiondeficit hyperactivity disorder (ADHD).

The method includes the step of administering to a subject in need oftreatment of a cognitive disorder a therapeutically effective amount ofa urolithin or a precursor thereof, to treat the cognitive disorder.

The method of the invention can be used alone or in combination with anymethod or compound known to be useful to treat a cognitive disorder. Forexample, In one embodiment, the method of the invention is combined withuse of a stimulant, such as methylphenidate (e.g., Ritalin®),dextroamphetamine (Dexedrine®), mixed amphetamine salts (Adderall®),dextromethamphetamine (Desoxyn®), and lisdexamphetamine (Vyvanase®).

An aspect of the invention is a method of treating or preventing astress-induced or stress-related cognitive dysfunction. As used herein,a “stress-induced or stress-related cognitive dysfunction” refers to adisturbance in cognitive function that is induced or related to stress.The method includes the step of administering to a subject in need oftreatment or prevention of a stress-induced or stress-related cognitivedysfunction a therapeutically effective amount of a urolithin or aprecursor thereof, to treat or prevent the stress-induced orstress-related cognitive dysfunction.

Mood Disorders

Brain tissue requires a high level of energy for its metabolism,including the maintenance of the transmembrane potential, signaltransduction and synaptic remodeling. An increase of psychiatricsymptoms and disorders, in particular depression, is likely present inpatients with mitochondrial disorders.

Mitochondrial structure and function, measured by a variety of differenttechniques, have been shown to be abnormal in patients with mooddisorders, including major depression as well as in the other affectivespectrum disorders.

Two studies revealed that a several-fold increased likelihood ofdeveloping depression can be maternally inherited along with the mtDNA,which strongly argues that mtDNA sequence variants may inducemitochondrial dysfunction that can predispose individuals towards thedevelopment of depression (Boles et al., 2005; Burnett et al., 2005).

The relationship between mitochondrial dysfunction and unipolardepression has been explored in several studies. In studies ofpostmortem brain from subjects with probable or diagnosed majordepression, of whom most subjects were (probably) medicated, no increaseof the common 5 kb mtDNA deletion could be detected (Kato et al., 1997;Sabunciyan et al., 2007; Shao et al., 2008 Stine et al., 1993).Alterations of translational products linked to mitochondrial functionwere found in the frontal, prefrontal and tertiary visual cortices(Karry et al., 2004; Whatley et al., 1996). Alterations of fourmitochondrial located proteins in the anterior cingulate cortex havebeen reported (Beasley et al., 2006). Decreased gene expression for 6 of13 mtDNA-encoded transcripts in frontal cortex tissue (Brodmann areas(BA) 9 and 46) (Shao et al., 2008), and of nDNA-encoded mitochondrialmRNA and proteins in the cerebellum, have also been reported in majordepression (Ben-Shachar and Karry, 2008). Levels of an electrontransport chain complex I subunit (NDUFS7), and complex I activity, inpostmortem prefrontal cortex were found to be below or at the lowestrange of the normal controls in half of the cases of major depressivedisorder in a recent study (Andreazza et al., 2010). In the two latterstudies, the authors were unable to detect any effect of medication onthe results.

Decreases of respiratory chain enzyme ratios and ATP production rates,and an increased prevalence of small mtDNA deletions (but not of thecommon 5 kb mtDNA deletion), were found in muscle from patients with alifetime diagnosis of major unipolar depression with concomitantphysical symptoms. Medication did not seem to influence the results(Gardner et al., 2003b). Clinical relevance was suggested by the findingthat essentially every depressed subject with very high degrees ofsomatic complaints demonstrated low ATP production rates in biopsiedmuscle (Gardner and Boles, 2008a).

An aspect of the invention is a method of treating a mood disorder (alsoknown as an affective disorder). As used herein, a “mood disorder”refers to a disturbance in emotional state, such as is set forth in theDiagnostic and Statistical Manual of Mental Disorders, published by theAmerican Psychiatric Association. Mood disorders include but are notlimited to major depression, postpartum depression, dysthymia, andbipolar disorder. In one embodiment, the mood disorder is majordepression.

The method includes the step of administering to a subject in need oftreatment of a mood disorder a therapeutically effective amount of aurolithin or a precursor thereof, to treat the mood disorder.

The method of the invention can be used alone or in combination with anymethod or compound known to be useful to treat a mood disorder. Forexample, In one embodiment, the method of the invention is combined withuse of an antidepressant agent. Antidepressant agents are well known inthe art and include selective serotonin reuptake inhibitors (SSRIs),serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic andspecific serotonergic antidepressants, norepinephrine reuptakeinhibitors, norepinephrine-dopamine reuptake inhibitors, selectiveserotonin reuptake inhibitors, norepinephrine-dopamine disinhibitors,tricyclic antidepressants, and monamine oxidase inhibitors.

An aspect of the invention is a method of treating or preventing astress-induced or stress-related mood disorder. As used herein, a“stress-induced or stress-related mood disorder” refers to a disturbancein emotional state that is induced or related to stress. Such mooddisorders are sometimes referred to as reactive mood disorders and areto be distinguished from other mood disorders, e.g., so-called organicmood disorders. The method includes the step of administering to asubject in need of treatment or prevention of a stress-induced orstress-related mood disorder an effective amount of a urolithin or aprecursor thereof, to treat or prevent the stress-induced orstress-related mood disorder.

An aspect of the invention is a method of treating an anxiety disorder.As used herein, an “anxiety disorder” refers to a dysfunctional state offear and anxiety, e.g., fear and anxiety that is out of proportion to astressful situation or the anticipation of a stressful situation. In oneembodiment, an anxiety disorder is any one or combination of generalizedanxiety disorder, panic disorder, panic disorder with agoraphobia,agoraphobia, social anxiety disorder, obsessive-compulsive disorder, andpost-traumatic stress disorder. In one embodiment, an anxiety disorderis any one or combination of generalized anxiety disorder,obsessive-compulsive disorder, panic disorder, post-traumatic stressdisorder, and social anxiety disorder. In one embodiment, an anxietydisorder is generalized stress disorder. In one embodiment, an anxietydisorder is post-traumatic stress disorder. In one embodiment, ananxiety disorder is a stress-induced anxiety disorder.

The method includes the step of administering to a subject in need oftreatment of an anxiety disorder a therapeutically effective amount of aurolithin or a precursor thereof, to treat the anxiety disorder.

The method of the invention can be used alone or in combination with anymethod or compound known to be useful to treat an anxiety disorder. Forexample, In one embodiment, the method of the invention is combined withuse of any one or combination of psychotherapy, benzodiazepines,buspirone (Buspar®), or beta-blockers. Benzodiazepines are well known inthe art and include, without limitation, clonazepam (Klonopin®),lorazepam (Ativan®), and alprazolam (Xanax®). Additional drugs that maybe used in combination with the methods of the invention includeimipramine (Tofranil®) and venlafaxine (Effexor®).

An aspect of the invention is a method of treating or preventing astress-induced or stress-related anxiety disorder. As used herein, a“stress-induced or stress-related anxiety disorder” refers to adysfunctional state of fear and anxiety that is induced or related tostress. Such anxiety disorders are sometimes referred to as reactiveanxiety disorders and are to be distinguished from other anxietydisorders, e.g., so-called organic anxiety disorders. The methodincludes the step of administering to a subject in need of treatment orprevention of a stress-induced or stress-related anxiety disorder aneffective amount of a urolithin or a precursor thereof, to treat orprevent the stress-induced or stress-related anxiety disorder.

An aspect of the invention is a method of promoting neurite outgrowth.In one embodiment, the method is an in vitro method. In one embodiment,the method is an in vivo method. As used herein, a “neurite” refers toany projection from the cell body of a neuron. In one embodiment, suchprojection is an axon. In one embodiment, such projection is a dendrite.The term is frequently used when speaking of immature or developingneurons, especially of cells in culture, because it can be difficult totell axons from dendrites before differentiation is complete. Neuritesare often packed with microtubule bundles, the growth of which isstimulated by nerve growth factor (NGF), as well as tau proteins,microtubule associated protein 1 (MAP1), and microtubule associatedprotein 2 (MAP2). The neural cell adhesion molecule N-CAM simultaneouslycombines with another N-CAM and a fibroblast growth factor receptor tostimulate the tyrosine kinase activity of that receptor to induce thegrowth of neurites.

Neurite outgrowth can be measured morphologically or functionally.Morphological measurement typically entails microscopic examination withmeasurement of the length and/or number of neurites.

As used herein, “promoting” refers to enhancing or inducing. In oneembodiment, “promoting” means inducing. For example, neurite outgrowthin a negative control sample may be negligible, while neurite outgrowthin an experimental or treatment sample may be non-negligible. In oneembodiment, “promoting” means enhancing. For example, neurite outgrowthin a negative control sample may be non-negligible, while neuriteoutgrowth in an experimental or treatment sample may be statisticallysignificantly greater than the negative control. Of course “promoting”as used herein can encompass both enhancing and inducing.

In one embodiment, the method includes the step of contacting a nervecell with an effective amount of a urolithin or a precursor thereof, topromote neurite outgrowth.

In one embodiment, the method includes the step of administering to asubject in need thereof a therapeutically effective amount of theurolithin or precursor thereof, to promote neurite outgrowth.

The methods of the invention can be used alone or in combination withany method or compound known to be useful to promote neurite outgrowth.For example, In one embodiment, a method of the invention can becombined with use of any one or more of NGF, tau protein, MAP1, MAP2,N-CAM, or an agent that induces the expression of any one or more ofNGF, tau protein, MAP1, MAP2, N-CAM, or fibroblast growth factorreceptor.

Using Neuronal Cells In Vitro to Screen Compounds for NeuroprotectiveActivities

In the processes of aging and neurodegeneration, the progressivedeterioration of cognitive function is essentially due to the loss ofentities sustaining neuronal communication. These entities areessentially composed of neuronal cell bodies, neurites and synapticcontacts that connect them to target cells. Neurons display very complexmorphologies. The most complex neuronal cell types, such as motorneurons extending axonal processes up to one meter long or nigraldopaminergic neurons making more than 150,000 synaptic contacts, willoften be the most vulnerable in normal aging or disease. To maintainsuch a complex architecture and effectively convey electrical andneurochemical signals, neurons heavily rely on energy supply. Therefore,axonal transport, synaptic activity, and the maintenance of iongradients highly depend on mitochondrial function. To carry on thesedemanding cellular functions, neurons experience over time difficultiesto sustain the delicate balance of mitochondrial activity and ensuingoxidative stress. Such imbalance is often considered the cause ofneuronal dysfunction or premature degeneration.

Therefore, any treatment promoting neuronal survival, or the formationof the neuronal processes and synaptic contacts that build up neuronalcomplex architecture, is expected to positively impact neuronalfunctions. The measurement of compound effects on neuronal functiontypically relies on the tedious monitoring of animal behavioraloutcomes, which is not amenable to medium- or high-throughput screeningof biological activities. In vitro models based on neuroblastoma celllines or primary neuronal cultures represent an accepted proxy to assesscompound effects on crucial morphological parameters, which will reflectthe ability of neurons to sustain their normal function in the mammalianbrain. Indicators such as the number of processes, their length, orcomplexity will reveal compound effects on critical steps ofintracellular signaling. Although one should keep in mind that suchparameters only indirectly reflect the performance of higher brainfunctions, they provide a valuable appraisal of compound efficacy thatmay translate into improved cognitive or motor functions in normal ordiseased conditions.

Metabolic Disorders

Mitochondrial function in key metabolic tissues (liver, muscle, adiposetissue, pancreas) is involved in the pathogenesis of metabolic diseases.In each of these tissues, mitochondrial oxidative activity must beappropriate to fully oxidize nutrient loads, particularly fatty acids.Failure of complete oxidation can lead to accumulation of lipidintermediates, incomplete fatty acid oxidation products, and ROS.Altogether, these cellular events contribute to fat accumulation,insulin resistance, altered insulin secretion, low grade inflammation,and oxidative stress, which are all components of type II diabetesmellitus and obesity.

The importance of mitochondrial activity in the pathogenesis ofmetabolic diseases has been established in several studies in humans.For example, insulin resistance in skeletal muscle has been associatedwith a defect in mitochondrial oxidative phosphorylation, where a 30%reduction in mitochondrial activity is observed in insulin-resistantoffspring of patients with type 2 diabetes as compared to controlsubjects. Petersen K F, et al. (2004) New Engl J Med. 350:664-71. It hasalso been observed that obese patients display a 20% decrease inmitochondrial activity along with a 35% reduction in mitochondrial sizecompared to healthy lean subjects. Petersen K F, et al. (2003) Science300:1140-2. Finally, age-associated decline in mitochondrial functioncontributes to insulin resistance in the elderly. Accordingly, a 40%reduction in mitochondrial oxidative and phosphorylation activity hasbeen reported in the elderly compared to young subjects. Theseobservations link disturbance in mitochondrial function to metabolicdisorders, especially “diabesity” (Kelley D E, et al. (2002) Diabetes51:2944-50).

Mitochondrial oxidative activity, also referred to as oxidativephosphorylation, can be considered as a key determinant underlying therisk of metabolic diseases. Reductions in mitochondrial activity can bemediated by genetic factors (e.g., family history, ethnicity),epigenetic mechanisms, developmental exposures, eating behavior andaging.

When sustained fuel excess (e.g., from overeating or impaired fatstorage) surpasses energetic demands and/or oxidative capacity, and/orappropriate compensatory mechanisms are insufficient (for example, dueto inactivity or failure of mitochondria to adapt to higher cellularoxidative demands), there is an increased risk of metabolic disorders.The resulting lipid accumulation and oxidative stress can altertranscriptional responses and damage mitochondria, further reducingoxidative phosphorylation capacity, compounding the deleterious effectsof fuel excess elevating the risk of metabolic disorders.

The sufficiency fully to oxidize fatty acids resides in the balancebetween: (i) the net mitochondrial oxidative activity (determined by theneed to generate energy to meet cellular demands, e.g., contraction andion transport), and (ii) fuel availability (determined by food intake,adiposity, and adipose storage capacity). Balance is achieved whenoxidative activity equals or exceeds fuel loads.

Under normal homeostatic conditions, both oxidative activity andcellular fuel availability can be altered to ensure that mitochondrialfunction is appropriate for the ambient metabolic environment. Forexample, cellular demand for energy can be increased through exercise,and fuel availability can be reduced through weight loss and/or reducedfood intake. In this context, inter-individual variations in oxidativecapacity and/or activity, fuel load, or ability to modulatemitochondrial activity (acute response), increase mitochondrial capacity(chronic response), or resolve oxidative stress could determine the setpoint of metabolic balance. Such differences could become prominentparticularly in an obesogenic environment (one characterized byenvironments that promote increased overall food intake, intake ofnonhealthful foods, and physical inactivity). Therefore, individualswith a high oxidative capacity or adaptive responses would have hightolerance to large fuel loads. Conversely, individuals with reducedoxidative capacity and/or suboptimal adaptive responses would beintolerant to moderate high fuel loads, leading to lipid accumulation,incomplete oxidation, production of ROS, and insulin resistance.

With time, insufficient compensation will result in chronic insulinresistance and metabolic disorders. Insufficient oxidative capacitycould be resolved by compensatory mechanisms that increase oxidativecapacity (e.g., exercise) or decrease fuel load (weight loss). However,these lifestyle changes appear usually insufficient or not achievablefor most overweight/obese and type 2 diabetic or pre-diabetic subjects.

Mitochondria are particularly important for skeletal muscle function,given the high oxidative demands imposed on this tissue by intermittentcontraction. Mitochondria play a critical role in ensuring adequatelevels of ATP needed for contraction by the muscle sarcomere. Thishigh-level requirement for ATP by sarcomeres has likely contributed tothe distinct subsarcolemmal and sarcomere-associated populations ofmitochondria in muscle. Moreover, muscle cells must maintain metabolicflexibility, the ability to rapidly modulate substrate oxidation as afunction of ambient hormonal and energetic conditions. For example,healthy muscle tissue predominantly oxidizes lipid in the fasting state,as evidenced by low respiratory quotient (RQ), with subsequenttransition to carbohydrate oxidation (increased RQ) during the fedstate. Availability of fuels, particularly lipids, and capacity tooxidize them within mitochondria are also critical for sustainedexercise. Thus, mitochondrial functional capacity is likely to directlyaffect muscle metabolic function and, because of its large contributionto total body mass, to have a significant impact on whole-bodymetabolism. This possibility is supported by the findings of increasedmitochondrial content in skeletal muscle in an individual withhypermetabolism and resistance to weight gain (Luft syndrome).

Insulin Resistance and Diabetes Mellitus

Skeletal muscle is the largest insulin-sensitive organ in humans,accounting for more than 80% of insulin-stimulated glucose disposal.Thus, insulin resistance in this tissue has a major impact on whole-bodyglucose homeostasis. Indeed, multiple metabolic defects have beenobserved in muscle from insulin-resistant but normoglycemic subjects athigh risk for diabetes development, including: (i) reducedinsulin-stimulated glycogen synthesis; (ii) alterations in insulinsignal transduction; and (iii) increased muscle lipid accumulation.Although it remains unclear at present whether any of these defects playa causal role in insulin resistance, intramyocellular lipid excessstrongly correlates with the severity of insulin resistance, even aftercorrection for the degree of obesity, and has been observed in musclesof multiple fiber types. Moreover, lipid excess has been linkedexperimentally to induction of insulin resistance and alterations ininsulin signal transduction. Thus, one possible mechanism by whichimpaired mitochondrial function might contribute to insulin resistanceis via altered metabolism of fatty acids. Increased tissue lipid load,as with obesity, and/or sustained inactivity, may lead to theaccumulation of fatty acyl coenzyme A (CoA), diacylglycerols, ceramides,products of incomplete oxidation, and ROS, all of which have been linkedexperimentally to reduced insulin signaling and action. Additionalmechanisms potentially linking impaired mitochondrial oxidative functionto insulin resistance include: (i) reduced ATP synthesis forenergy-requiring functions such as insulin-stimulated glucose uptake;(ii) abnormalities in calcium homeostasis (necessary forexercise-induced glucose uptake); and (iii) reduced ATP productionduring exercise, potentially contributing to reduced aerobic capacity,muscle fatigue, and decreased voluntary exercise over time—furtherfeeding a vicious cycle of inactivity-fueled insulin resistance.

Mitochondrial capacity is central to the key function of the pancreaticbeta ((3)-cell-regulated insulin secretion. Both rapid (first phase) andmore prolonged (second phase) insulin secretion are dependent on glucosemetabolism and mitochondrial oxidative capacity; glucose oxidationincreases the ATP/ADP ratio, inhibiting plasma membrane K-ATP channelsand allowing voltage-gated calcium channels to open. Increasedcytoplasmic calcium then triggers exocytosis of plasma-membrane dockedinsulin granules (first phase). Subsequent recruitment of granules tothe plasma membrane (second phase) appears to depend on mitochondrialmetabolites produced by anaplerosis. Mitochondrial metabolism is alsorequired for the transient, controlled production of ROS, which isrequired for the mitochondrial signaling pathways that trigger granuleexocytosis.

Mitochondrial diabetes only develops upon aging, with an average age ofonset between 35 and 40 yr for maternally inherited diabetes withdeafness (MIDD) and 48 yr for 14577 T/C, a mitochondrial DNA missensemutation in maternally inherited type 2 diabetes. This contrasts withthe early childhood onset of diabetes in syndromes such asmaturity-onset diabetes of the young 2 (MODY2), in which a mutation inglucokinase, the first step of glycolysis, results in attenuatedglucose-stimulated ATP generation and insulin secretion. These datasuggest that mitochondrial diabetes is more likely to result from agradual deterioration of n-cell function, rather than from an acutefunctional impairment due to insufficient ATP production.

Mitochondrial function in tissues involved in the pathogenesis ofdiabetes mellitus (liver, muscle, adipose tissue, and pancreaticβ-cells) is critical for multiple aspects of cellular metabolism. Ineach of these tissues, mitochondrial oxidative activity must beappropriate to fully oxidize nutrient loads, particularly fatty acids.Failure of complete oxidation can lead to accumulation of lipidintermediates, incomplete fatty acid oxidation products, and ROS,inducing both insulin resistance (muscle, liver, adipose) and alteredsecretion (β-cells).

Mild deficiencies in mitochondrial activity, and/or an inability toincrease activity and capacity in response to cellular energy demand,could explain the reduced exercise ability seen in individuals with afamily history of diabetes mellitus. Over time, this phenotype couldcontribute to reduced voluntary exercise and increase the likelihood ofan imbalance between mitochondrial activity and fatty acid load.Secondly, chronic imbalance in energy metabolism due to overnutrition,obesity, and inactivity could directly contribute to increased cellularand mitochondrial ROS production. In turn, excessive ROS can induce bothinsulin resistance and mitochondrial dysfunction. For example, ahigh-fat, high-sucrose diet in the diabetes-prone C57BL6 mouse causesmitochondrial alterations in parallel with enhanced ROS production andimpaired insulin sensitivity. Similarly, exposure of muscle cells invitro to saturated fatty acids or high-fat feeding in mice results inalterations in mitochondrial structure and insulin resistance, both ofwhich are reversed by antioxidants. Thus, oxidative stress can inducemitochondrial dysfunction in parallel with insulin resistance—perhaps anadaptive response aimed at limiting further oxidative damage.Importantly, resolution of oxidative stress can reverse insulinresistance.

Muscle Performance

In other embodiments, the invention provides methods for enhancingmuscle performance by administering a therapeutically effective amountof a mitochondria-enhancing or -activating extract, formulation orcompound. For example, extracts containing ellagitannins or ellagicacid, or compositions containing ellagitannins, ellagic acid, orurolithins behave to activate mitochondria and may be useful forimproving physical endurance (e.g., ability to perform a physical tasksuch as exercise, physical labor, sports activities), inhibiting orretarding physical fatigue, enhancing blood oxygen levels, enhancingenergy in healthy individuals, enhancing working capacity and endurance,reducing muscle fatigue, reducing stress, enhancing cardiac andcardiovascular function, improving sexual ability, increasing muscle ATPlevels, and/or reducing lactic acid in blood. In certain embodiments,the methods involve administering an amount of an ellagitannin- orellagic acid-containing natural extract, or compositions containingellagitannins, ellagic acid or urolithin, that increase mitochondrialactivity, increase mitochondrial biogenesis, and/or increasemitochondrial mass.

Sports performance refers to the ability of an athlete's muscles toperform when participating in sports activities. Enhanced sportsperformance, strength, speed, and endurance are measured by an increasein muscular contraction strength, increase in amplitude of musclecontraction, or shortening of muscle reaction time between stimulationand contraction. Athlete refers to an individual who participates insports at any level and who seeks to achieve an improved level ofstrength, speed, or endurance in their performance, such as, forexample, body builders, bicyclists, long distance runners, and shortdistance runners. Enhanced sports performance is manifested by theability to overcome muscle fatigue, ability to maintain activity forlonger periods of time, and have a more effective workout.

It is contemplated that the compositions and methods of the presentinvention will also be effective in the treatment of muscle-relatedpathological conditions, including myopathies, neuromuscular diseases,such as Duchenne muscular dystrophy, acute sarcopenia, for example,muscle atrophy and/or cachexia associated with burns, bed rest, limbimmobilization, or major thoracic, abdominal, and/or orthopedic surgery.

Chronic Stress

Chronic stress has also been reported to have a significant effect oncognitive performance and more precisely on learning and memoryprocesses (Sandi 2004; Sandi and Pinelo-Nava 2007). Several factors aredeterminant on the impact that chronic stress will have on cognitivefunction. The levels of stress are important in determining whether thestress will serve to facilitate cognitive function or be deleterious. Itis thought that in response to stressful situations the body inducesstress hormones, which produce an inverted U effect on learning, memory,and plasticity. Baldi et al. (2005) Nonlinearity Biol Toxicol Med. 3(1)9-21; Joels (2006) Trends Pharmacol Sci. 27(5):244-50. Thus the level ofstress has a great effect on cognitive function, with high levels ofstress resulting in high levels of stress hormones and decreasedperformance.

The length of the stress, chronic vs. acute, has been also shown to playan important role, with distinct effects on cognitive function, as wellas brain structure and function (Sandi and Loscertales 1999; Pinnock andHerbert 2001). Also, stress acts on the memory forming process resultingin different outcomes, with consolidation (memory storage) beingfacilitated by acute stress, and retrieval (memory recall) beinginhibited (Roozendaal 2003). In addition, the predictability of thestress also plays a role on the severity of the effects observed oncognitive performance (Maier and Watkins 2005).

Additionally, the context in which the chronic stress occurs, as well asindividual differences in stress response inherent to individuals andgender, play an important role in determining the final cognitive impactof chronic stress (Bowman, Beck et al. 2003; Shors 2004; Joels, Pu etal. 2006).

The biological basis for the effects of chronic stress is not yet welldefined. However, a common observed feature is the key role ofglucocorticoids in mediating both, the facilitating and impairingactions of stress, on different memory processes and phases. While themechanism of glucocorticoids action has yet to be elucidated, it hasbeen shown in vitro to impair neuronal outgrowth induced by nerve growthfactor (NGF). Unsicker et al. (1978) Proc Natl Acad Sci USA.75:3498-502. Furthermore, neuronal structure and neurite outgrowthinduced by factors such as NGF correlate strongly with theirneuroprotective activity, suggesting again that neuronal structure isimportant for cognition.

Stress and Structural Remodeling

Initially, the hippocampus was the brain region that received closeattention due to the many reports indicating impairing effects ofchronic stress in hippocampus-dependent memory tasks. However, intensivework is now providing evidence for a more integral impact of chronicstress throughout the brain, with major changes having also beingreported for the prefrontal cortex and the amygdala. Changes indendritic branching and synaptogenesis occurring in the amygdala areplausible candidates to participate in stress-induced mood alterations.Also, changes occurring at the level of the hippocampus and theprefrontal cortex are believed to play a key role in stress-induced moodalterations.

Hippocampus.

The hippocampus is well known for its crucial role in memory processes.Hippocampus-dependent tasks are generally affected by both acute andchronic stress manipulations. In humans, neuroimaging studies havereported hippocampal atrophy in association with stress- andglucocorticoid-related cognitive and neuropsychiatric alterations,including depression.

In rodents, a prominent and many times replicated effect is a dendriticatrophy in apical dendrites from CA3 pyramidal neurons. This reduceddendritic branching has been associated with (i) a reduction in synapticdensity of excitatory glutamatergic synapses; (ii) a shrinkage of thevolume of the complex dendritic spines termed dendritic excrescences,that are located on the proximal apical dendrite and soma of CA3pyramidal cells and which serve as postsynaptic targets for the mossyfiber synaptic inputs; and (iii) a rearrangement of synaptic vesiclesand mitochondria in the afferent mossy fiber terminals. On its turn,evidence for synaptic remodeling—in terms of changes in synapticfeatures—has also been reported for the hippocampal CAl region.

Prefrontal Cortex.

The prefrontal cortex (PFC), and more particularly its medial part(mPFC), plays key roles in higher cognitive processes (includingexecutive function, working memory, attention), as well as in theintegration of cognitive and emotionally relevant information. It shouldbe noted that the mPFC contains high levels of glucocorticoid receptorsand is involved in the regulation of stress-inducedhypothalamic-pituitary-adrenal (HPA) activity. As noted above, clinicalevidence highlights the mPFC as an area that experiences markedalterations in a wide variety of neuropsychiatric disorders, includingdepression.

There is substantial evidence from rodent studies for stress-induceddendritic shrinkage in the PFC. In particular, major neuronal remodelingwas described to occur in layer II/III of the mPFC as a consequence ofrepeated exposure to chronic stress or repeated glucocorticoidtreatment. The major described changes in this area are (i) a dendriticatrophy, including both decrease of total length and number of apicaldendrites from pyramidal neurons; and (ii) a decrease in apicaldendritic spine density (approximately one-third of all axospinoussynapses on apical dendrites of pyramidal neurons are lost).

Antidepressant Effects.

Treatment with the atypical (modified tricyclic) antidepressanttianeptine was shown to reverse dendritic atrophy induced by chronicstress in CA3 pyramidal neurons in rats. Moreover, antidepressants werealso reported to facilitate axonal and dendritic sprouting. Thesefindings suggest that antidepressants can have a major impact onneuronal remodeling, providing the basis for relevant circuits to bereorganized in the course of recovery from depression.

Early-Life Stress

An aspect of the invention is a method for treating mood effects ofearly-life stress. The method includes the step of administering to asubject in need thereof a therapeutically effective amount of urolithinor a precursor thereof, to treat the effects of early-life stress onmood, depression, anxiety, and risk-taking behavior.

Early-life stress has been reported to have a significant detrimentaleffect on cognitive performance, including psychological parameters suchas increased rates of or susceptibility to depression, anxiety, andabnormal risk-taking behavior. Heim C, Nemeroff C B. (2001) BiolPsychiatry 49:1023-1039. Increased rates ofattention-deficit/hyperactivity disorder (ADHD), post-traumatic stressdisorder (PTSD), and major depression have been reported in individualshaving experienced early-life stress. Famularo R et al. (1992) J Am AcadChild Adolesc Psychiatry 31:863-867; Pelcovitz D et al. (1994) J Am AcadChild Adolesc Psychiatry 33:305-312. Early-life stress is thought tohave an impact on the hypothalamic-pituitary-adrenal (HPA) axis. Ladd CO et al. (2000) Prog Brain Res 122:81-103. The key effector thought tocontrol the responsiveness of the HPA axis to stress is the centralcorticotrophin releasing factors (CRF).

CRF is a 41 amino acid peptide which is distributed throughout the CNS.This includes the cell bodies of the medial parvocellular region of thehypothalamic paraventricular nucleus (PVN), a central component of theHPA axis. Upon stress, CRF is released from the median eminence nerveterminals into the hypothalamo-hypophysial portal circulation andtransported to the anterior pituitary where it binds to CRF receptors(CRF1 and CRF2). CRF binding to the CRF1 receptor produces effects thatare reminiscent of stress, depression, and anxiety. CRF binding to CRF2receptor stimulates the production and release of adrenocorticotropichormone (ACTH), which in turn stimulates the production ofglucocorticoids involved in the stress response.

In models of early-life stress caused by maternal separation, aconsistent long-term elevated level of CRF mRNA is observed. Plotsky P Met al. (2005) Neuropsychopharmacology 30:2192-2204. Such increases inCRF have been shown to have effects at the level of amygdala inincreasing anxiety response. It is thought that a persistentsensitization of the CRF neurocircuits is responsible for the abnormallyelevated anxiety, depression, and risk-taking behavior observed in miceexposed to early-life stress.

Current Strategies Employing Antidepressants to Improve PsychologicalDisorders Due to Early-Life Stress

A number of studies have shown that antidepressants decrease CRFactivity in the HPA axis in rodents and primates, including humans.Banki C M et al. (1992) J Affect Disord 25:39-45; Brady L S et al.(1992) Brain Res 572:117-125; Brady L S et al. (1991) J Clin Invest87:831-837; De Bellis M D et al. (1993) Am J Psychiatry 150:656-657;Veith R C et al. (1993) Psychiatry Res 46:1-8. Several classes ofantidepressant drugs appear to produce a decrease in the activity of oneor more CRF neural systems. These include selective 5-HT reuptakeinhibitors (SSRI), which have been shown to be effective in thetreatment of several psychiatric disorders that have been associatedwith early-life stress (e.g., depression and PTSD). Hidalgo R B et al.(2000) J Psychopharmacol 14:70-76. Notably, in a randomizedplacebo-controlled trial, subjects having undergone early-life stressand suffering from PTSD were responsive to fluoxetine. van der Kolk B Aet al. (1994) J Clin Psychiatry 55:517-522. Furthermore, SSRIs,including fluoxetine and paroxetine, show significant efficacy versusplacebo in the treatment of early-onset depression in children andadolescents. Martin A et al. (2000) Child Adolesc Psychiatr Clin N Am9:135-157. Tricyclic antidepressants have also been found to reverseincreased HPA axis reactivity to stress in adult primates exposed tomaternal deprivation. Suomi S J. (1991) Ciba Found Symp 156:171-183. Itappears that several available drugs, including the SSRIs, may bebeneficial in the treatment of children and adults exposed to early-lifestress. Fisher P A et al. (2000) J Am Acad Child Adolesc Psychiatry39:1356-1364.

Additional Indications

The invention will also find use in the treatment of any of a variety ofadditional diseases and conditions in which defective or diminishedmitochondrial activity participates in the pathophysiology of thedisease or condition, or in which increased mitochondrial function willyield a desired beneficial effect. As an example, the invention furtherincludes methods and compounds that may be used to treat maleinfertility associated with diminished sperm motility. Nakada et al.(2006) Proc Natl Acad Sci USA. 103:15148-53. As another example, theinvention further includes methods and compounds that may be used totreat macular degeneration and certain other age-related and inheritedeye disorders. Khandhadia et al. (2010) Expert Rev Mol Med. 12: e34;Jarrett et al. (2010) Ophthalmic Res. 44:179-90. Another example is amethod of treating hearing loss, including but not limited toage-related hearing loss. In each of these and other indications, themethod involves administering to a subject in need of such treatment aneffective amount of a urolithin or precursor thereof, as disclosedherein, to treat the indication.

Formulations and Clinical Use

A “subject” as used herein refers to a living vertebrate. In oneembodiment, a subject is a mammal. In one embodiment, a subject is ahuman.

As used herein, the term “treat” as used in connection with a disease,disorder, or condition of a subject, means to reduce by a detectableamount at least one clinical or objective manifestation of the disease,disorder, or condition of a subject. In one embodiment, the term “treat”used in connection with a disease, disorder, or condition of a subject,means to cure the disease, disorder, or condition of a subject.

The urolithin or precursor thereof may be administered, alone ortogether with another agent, to a subject (e.g., mammal) in a variety ofways. For example, the urolithin or precursor thereof can beadministered orally or parenterally. Parenterally includes, withoutlimitation, intravenously, intramuscularly, intraperitoneally,subcutaneously, intra-articularly, intrasynovially, intraocularly,intrathecally, topically, or by inhalation. As such, the form of theurolithin or precursor thereof dose can be in a variety of forms,including natural foods, processed foods, natural juices, concentratesand extracts, injectable solutions, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, nosedrops, eyedrops,sublingual tablets, and sustained-release preparations.

The compounds of this invention can be provided in isolated form. Asused herein, the term “isolated” means substantially removed from othercompounds or components with which the compound of interest mayotherwise be found, for example, as found in nature. In one embodiment,a compound is isolated when it is essentially completely removed fromother compounds or components with which the compound of interest mayotherwise be found. In one embodiment, a compound is isolated when it ispure.

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate pharmaceutically acceptablecarriers or diluents, and may be formulated into preparations in solid,semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. As such, administration of thecompounds can be achieved in various ways, including oral, buccal,rectal, parenteral, intraperitoneal, intradermal, transdermal, andintratracheal administration. The active agent may be systemic afteradministration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation.

The compounds of the invention can also be formulated as food additives,food ingredients, functional foods, dietary supplements, medical foods,nutraceuticals, or food supplements.

In pharmaceutical dosage forms, the compounds may be administered in theform of their pharmaceutically acceptable salts. They may also be usedin appropriate association with other pharmaceutically active compounds.The following methods and excipients are merely exemplary and are in noway limiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional, additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier, wherein each dosageunit, for example, mL or L, contains a predetermined amount of thecomposition containing one or more compounds of the present invention.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres; slabs, etc., with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing the inhibitory compounds may be placedin proximity to a site of interest, so that the local concentration ofactive agent is increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to, be achieved, and the pharmacodynamicsassociated with each compound in the host

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

For clinical use, the urolithin or urolithin precursor is administeredin a therapeutically effective amount. As used herein, an “effectiveamount” refers to an amount that is sufficient to realize a desiredbiological effect. As used herein, a “therapeutically effective amount”refers to an amount sufficient to realize, in a single dose or multipledoses, a desired therapeutic effect. A skilled artisan can determinetherapeutically effective amounts based on in vitro, preclinical, orclinical studies, or any combination thereof.

Dosing will generally be daily to weekly. In one embodiment, dosing isat least weekly. For example, a subject may receive one dose onceweekly, twice weekly, thrice weekly, or every other day. In oneembodiment, dosing is at least daily. For example, a subject may receiveone or more doses daily.

For clinical use, a urolithin will generally be administered in anamount ranging from about to 0.2-150 milligram (mg) of urolithin perkilogram (kg) of body weight of the subject. In one embodiment, theurolithin or precursor thereof is administered in a dose equal orequivalent to 2-120 mg of urolithin per kg body weight of the subject.In one embodiment, the urolithin or precursor thereof is administered ina dose equal or equivalent to 4-90 mg of urolithin per kg body weight ofthe subject. In one embodiment, the urolithin or precursor thereof isadministered in a dose equal or equivalent to 8-30 mg of urolithin perkg body weight of the subject. Where a precursor of urolithin is to beadministered rather than a urolithin, it is administered in an amountthat is equivalent to the above-stated amounts of urolithin.

Any given dose may be given as a single dose or as divided doses.

In one embodiment, the urolithin or precursor thereof is administered ina dose sufficient to achieve a peak serum level of at least 0.001micromolar (μM). In one embodiment, the urolithin or precursor thereofis administered in a dose sufficient to achieve a peak serum level of atleast 0.01 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 0.1 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 1 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 5 μM. In one embodiment, the urolithin or precursor thereof isadministered in a dose sufficient to achieve a peak serum level of atleast 10 μM.

In one embodiment, the urolithin or precursor thereof is administered ina dose sufficient to achieve a sustained serum level of at least 0.001micromolar (μM). In one embodiment, the urolithin or precursor thereofis administered in a dose sufficient to achieve a sustained serum levelof at least 0.01 μM. In one embodiment, the urolithin or precursorthereof is administered in a dose sufficient to achieve a sustainedserum level of at least 0.1 μM. In one embodiment, the urolithin orprecursor thereof is administered in a dose sufficient to achieve asustained serum level of at least 1 μM. In one embodiment, the urolithinor precursor thereof is administered in a dose sufficient to achieve asustained serum level of at least 5 μM. In one embodiment, the urolithinor precursor thereof is administered in a dose sufficient to achieve asustained serum level of at least 10 μM. The sustained serum level canbe measured using any suitable method, for example, high pressure liquidchromatography (HPLC) or HPLC-MS.

In one embodiment, the urolithin or precursor thereof is administered aspomegranate juice in the amount of 25 mL to 5 L, or an equivalent doseof ellagitannins, ellagic acid, urolithins, or any combination thereof.Table 4 shows the consumption of different pomegranate compounds fordifferent levels of pomegranate juice. The range covers differences incompound concentration among different varieties of pomegranate. For thecalculations of ellagic acid equivalents, it was assumed that themetabolism of each mole of punicalagin resulted in the release of 1 moleof ellagic acid, and that this conversion happened with 100% efficiency.Levels of urolithin were determined by assuming that all the ellagicacid present, including that derived from punicalagin, converted tourolithin with 100% efficiency. Not taken into consideration were othersources of ellagic acid besides punicalagin and ellagic acid.

TABLE 4 Juice Ellagic Acid Total Equivalent Punicalagin Ellagic AcidEquivalents Urolithin (mL) (mg/d) (mg/d) (1:1) (mg/d) (1:1) (mg/d) 2510-65  3-30 20-35 15-25 50 20-130 6-60 40-70 30-50 75 30-195 9-90 60-105 45-75 100 40-260 12-120  80-140  60-100 150 60-390 18-180120-210  90-150 200 80-510 24-240 160-280 120-200 250 100-650  30-300200-350 150-250 500 200-1300 60-600 400-700 300-500 750 300-1950 90-900 600-1050 450-750 1000 400-1600 120-1200  800-1400  600-1000 2000800-3200 240-2400 1600-2800 1200-2000 3000 1200-4800  360-3600 2400-42001800-3000 4000 1600-6400  480-4800 3200-5600 2400-4000 5000 2000-13000600-6000 4000-7000 3000-5000

In one embodiment, the subject is not taking a urolithin or precursorthereof for any purpose other than for the treatment of a condition inaccordance with the methods of the invention. In one embodiment, thesubject is not taking a urolithin or precursor thereof for the treatmentof atherosclerosis, thrombosis, cancer, unwanted angiogenesis,infection, or inflammation.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following, which is included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and is not intended to limit the invention.

Example 1 Preparation of Functional Extracts from Pomegranate Compounds

The pomegranate extracts described in this application containingspecific molecules were prepared using an extraction procedure based onadsorption of polyphenols in a standard polymer adsorption-based columnas described. For the preparation of the extracts 31008 and 1108 derivedfrom pomegranate juice, pomegranates were juiced using a standardjuicing and manufacturing process and adsorbed onto a polymericchromatographic resin as pure juice. The resin Amberlite XAD-16 (Rohm &Haas) was packed into semi-preparative columns and loaded with theextracted juice. The column was washed with water to remove the sugarsuntil completion (Brix levels were below 0.1%). The polyphenols wereeluted with 100% ethanol. The remaining ethanol was evaporated undervacuum to produce a concentrated extract containing 4.5 g of totalpolyphenol per liter as determined using the Folin assay for totalpolyphenol content. Extract 1011 was prepared in a similar manner asextract 31008 and 1108, but the liquid extract was then spray driedutilizing a spray dryer to produce a final powder extract. UtilizingHPLC-MS for the identification of compounds, extract 31008, 1108, and1011 were found to contain the molecules punicalagin, punicalin,tellimagrandin, and pedunculagin.

The extract 71109 derived from the pomegranate husk was prepared bymanually separating the husk from the pomegranate arils pulp, followedby pressing with a manual fruit press. To extract the maximal amount ofpolyphenols, the cake/pomace of pressed pomegranate parts were soaked inwater consecutively for several periods of time (5 minutes) in order toincrease extraction efficiency. The extracted pomegranate solution wasclarified by centrifugation before being adsorbed onto the polymericchromatographic Amberlite XAD-16 resin (Rohm & Haas), packed insemi-preparative columns, and loaded with the water extracted frompomegranate husk. The column was washed with water to remove the sugarsuntil completion (Brix levels were below 0.1%). The polyphenols wereeluted with 100% ethanol. The remaining ethanol was evaporated undervacuum to produce a concentrated extract containing 17.1 g of totalpolyphenol per liter as determined using the Folin assay for totalpolyphenol content. This technique is a modification of methods known inthe art as described by several published methods for purification ofpolyphenols from various plants and berries. Tuck, K. L. and P. J.Hayball (2002) “Major phenolic compounds in olive oil: metabolism andhealth effects.” J Nutr Biochem 13(11):636-644; and Schieber, A., P.Hilt, et al. (2003) “A new process for the combined recovery of pectinand phenolic compounds from apple pomace.” Innovative Food Sci. EmergingTechnol. 4:99-107.

For the preparation of Extract 61109, an aqueous extract of thepomegranate was fractionated utilizing centrifugal partitionchromatography. The isolation fractions were lyophylized to produceextract 61109, highly enriched in punicalagin (>90%).

Purification of Punicalagin Preparation of Extract

Extract from pomegranate was dissolved in 16 mL of the organic/aqueousphase mixture (1:1) and filtered on a Teflon filter (0.45 μm).

Separation of Punicalagin from Extract Using Centrifugal PartitionChromatography

Separation of punicalagin from pomegranate extract was achieved byutilizing Centrifugal Partition Chromatography CPC. The CPC apparatuswas a FCPC® 1000 apparatus provided by Kromaton Technologies (Angers,France) that is fitted with a rotor of 1000 mL capacity. The solventswere pumped by a 4-way binary high-pressure gradient pump. The sampleswere introduced into the CPC column via a high pressure injection valve(Rheodyne) equipped with a 20 mL sample loop. The effluent was monitoredwith a diode array detection (DAD) detector equipped with a preparativeflow cell. Fractions were collected by a fraction collector. Theseparation steps were conducted at room temperature.

To accomplish the extraction, the stationary phase was first introducedinto the column in the ascending mode without rotating, and mobile phasewas then pumped through the stationary phase until an equilibrium stagewas reached. Then, the rotation speed was increased from 0 to 1000 rpmand the mobile phase was pumped into the column at a flow-rate of 20mL/min. After injection of 10 g of pomegranate extract, fractions of 20mL were collected every minute. The content of the outgoing organicphase was monitored by online UV absorbance measurement at λ=260 nm.

An elution-extrusion procedure was used to recover all the compoundsfrom the column: after a classical elution of 100 min, the mobile phasewas replaced by the stationary phase used as mobile liquid, until allvolume contained (1000 mL) was pushed out the column. A fractioncontaining punicalagins (mixture of A and B isomers) with 94-97%chromatographic purity was obtained between 51 and 63 minutes ofelution, and a second fraction with a chromatographic purity of 85-88%was obtained between 64 and 79 min.

To determine the level of purification, the purified sample was examinedusing by HPLC-DAD at a detection wavelength of 260 nm. The sample wasrun over a Prosontil C18, 5 μm, 250×4 mm column. The solvents used wereH₂O mQ+9.1% TFA/Acetonitrile +0.1% TFA at a flow rate of 1 mL/min.

Example 2 In Vitro Screening Assays for Compounds Promoting Enhancementof the Expression of Mitochondrial Genes in a Prototypical SkeletalMuscle Cell Line (C2C12 Myotubes)

Skeletal muscles have a pivotal role in the regulation of metabolichomeostasis since they are involved in metabolic functions such asenergy expenditure and maintenance of insulin sensitivity. Thesefunctions are tightly linked to mitochondrial activity, and impairmentof mitochondrial function has a causal role in defective metabolichomeostasis and development of metabolic disorders such as type 2diabetes, obesity, and dyslipidemia. Gene expression profile of genesinvolved in mitochondrial activity in differentiated C2C12 cells(myotubes) is an appropriate model to assess the impact of compounds onmitochondrial activity by evaluating numerous pathways which reflectmitochondrial activity, e.g., mitochondrial biogenesis, glycolysis,fatty acid β-oxidation, electron transport chain (ETC), mitochondrialdynamics.

To assess the effects of compounds on mitochondrial gene expression,C2C12 myoblasts were differentiated into myotubes by serum deprivationfor 4 days (Canto et al. (2009) Nature. 458:1056-60). Myotubes wereincubated for 48 hr with ellagic acid or urolithin A at a finalconcentration of 1, 10 or 50 μM (all dissolved in DMSO, finalconcentration 0.1%). DMSO was used as a control (final concentration0.1%). At the end of the treatment, cells were washed with phosphatebuffered saline (PBS) and mRNA were immediately extracted according tomanufacturer's instructions (Trizol Reagent, Invitrogen) by adding 1 mLof Trizol reagent. After extraction, cDNA were produced by reversetranscription according to manufacturer's instructions.

Assessment of the expression levels of genes (PGC-1α, Tfam, PFKFB3,CPT1b, MCAD, LCAD, Ndufa2, Cyt c, and Mfn2) which control themitochondrial function was performed by real time quantitative PCR(Watanabe et al. (2004) J Clin Invest. 113:1408-18) by using thefollowing set of primers (Fwd: forward primer; Rev: reverse primer):

PGC-1α: (Fwd) (SEQ ID NO: 1) AAGTGTGGAACTCTCTGGAACTG (Rev)(SEQ ID NO: 2) GGGTTATCTTGGTTGGCTTTATG Tfam: (Fwd) (SEQ ID NO: 3)AAGTGTTTTTCCAGCATGGG (Rev) (SEQ ID NO: 4) GGCTGCAATTTTCCTAACCA PFKFB3:(Fwd) (SEQ ID NO: 5) TCATGGAATAGAGCGCC (Rev) (SEQ ID NO: 6)GTGTGCTCACCGATTCTACA CPT1b: (Fwd) (SEQ ID NO: 7) CCCATGTGCTCCTACCAGAT(Rev) (SEQ ID NO: 8) CCTTGAAGAAGCGACCTTTG MCAD: (Fwd) (SEQ ID NO: 9)GATCGCAATGGGTGCTTTTGATAGAA (Rev) (SEQ ID NO: 10)AGCTGATTGGCAATGTCTCCAGCAAA LCAD: (Fwd) (SEQ ID NO: 11)GTAGCTTATGAATGTGTGCAACTC (Rev) (SEQ ID NO: 12) GTCTTGCGATCAGCTCTTTCATTANdufa2: (Fwd) (SEQ ID NO: 13) GCACACATTTCCCCACACTG (Rev) (SEQ ID NO: 14)CCCAACCTGCCCATTCTGAT Cyt c: (Fwd) (SEQ ID NO: 15) TCCATCAGGGTATCCTCTCC(Rev) (SEQ ID NO: 16) GGAGGCAAGCATAAGACTGG Mfn2: (Fwd) (SEQ ID NO: 17)ACGTCAAAGGGTACCTGTCCA (Rev) (SEQ ID NO: 18) CAATCCCAGATGGCAGAACTT

PGC-1α (PPARy-coregulator 1α) and Tfam (mitochondrial transcriptionfactor A) are master regulators of the mitochondrial function, namely ofmitochondrial biogenesis and mitochondrial phosphorylative oxidation(mOXPHOS). An increase in their expression levels reveals an overallenhancement of mitochondrial activity. The assessment of other targetgenes involved in key functions of the mitochondria allows identifyingthe enhanced pathways. PFKFB3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) is a key enzymeof glycolysis, i.e., the use of glucose to produce energy. In aerobicconditions (i.e., when there are supplies of oxygen), pyruvate producedfrom glucose via glycolysis is used by the mitochondria to produceenergy (ATP) through the Krebs cycle. CPT1b (carnitineO-palmitoyltransferase 1b), MCAD (medium chain acyl CoA dehydrogenase),and LCAD (long chain acyl CoA dehydrogenase) play a pivotal role inmitochondrial fatty acid uptake and β-oxidation, two critical steps forenergy production from fatty acids. Ndufa2 (NADH dehydrogenase[ubiquinone] 1 alpha subcomplex subunit 2) and Cyt c (cytochrome c) aresubunits of complex I and IV of the mitochondrial electron transportchain, respectively. These proteins have an essential role inmitochondrial respiratory chain and energy production from reducedequivalent produced by the Krebs cycle. Mfn2 (Mitofusin 2) is involvedin mitochondrial dynamics and fusion process. Its expression isincreased in the context of increased mitochondrial remodeling and/ormitochondrial biogenesis (increased number of mitochondria per cell).

The data depicted in FIG. 3 clearly indicate that ellagic acid andurolithin A increase mitochondrial activity in a dose-dependent mannerby modulating the expression of numerous genes involved in severalpathways of mitochondrial metabolism.

Example 3 In Vitro Screening Assay for Compounds Promoting Enhancementof Mitochondrial Activity in a Prototypical Skeletal Muscle Cell Line(C2C12 Myotubes)

Citrate synthase is the initial enzyme of the tricarboxylic acid (TCA)cycle and the rate limiting step to enter the TCA cycle. The TCA cyclewill produce NADH2 and FADH2, which are then used to fuel the electrontransport chain which will generate a proton (energy) gradient, whichwill be used in the generation of ATP. As such, citrate synthase is anexclusive marker of the mitochondrial number and of the mitochondrialactivity. By measuring the effects of compounds or formulations oncitrate synthase enzyme activity, it is possible to assess the abilityof the compounds to stimulate mitochondria activity (i.e., OXPHOS andATP production).

The enzyme citrate synthase catalyzes the reaction between acetylcoenzyme A (acetyl CoA) and oxaloacetic acid to form citric acid. Theacetyl CoA contributes 2 carbons to the 4 carbons of oxaloacetate,resulting in citrate with 6 carbons. The hydrolysis of the thioester ofacetyl CoA results in the formation of CoA with a thiol group (CoA-SH).The activity of citrate synthase is measured via the reaction betweenthe thiol of CoA-SH with the DTNB in the mixture to form5-thio-2-nitrobenzoic acid (TNB). This yellow product (TNB) is observedspectrophotometrically by measuring absorbance at 412 nm (CitrateSynthase Assay Kit, Cat Number CS0720, Sigma Aldrich).

C2C12 myoblasts were differentiated into myotubes by serum deprivationfor 4 days (Canto et al. (2009) Nature. 458:1056-60). Myotubes wereincubated for 48 hr with punicalagin at a final concentration of 1 or 10μM or with ellagic acid or urolithin at a final concentration of 1, 10or 50 μM (all dissolved in DMSO, final concentration 0.1%). DMSO wasused as a control (final concentration 0.1%). At the end of thetreatment, cells were washed 3 times with PBS and assayed for citratesynthase activity according to manufacturer's instructions (CitrateSynthase Assay Kit, Cat Number CS0720, Sigma Aldrich).

As depicted in FIG. 4, punicalagin, ellagic acid, and urolithin increasecitrate synthase activity in a dose-dependent manner, illustrating anoverall increase in mitochondrial activity and/or mitochondrial density(number of mitochondria per cells). These results confirm the resultsobtained by gene expression profile of mitochondrial genes (Example 1)showing an enhancement of mitochondrial activity and mitochondrialbiogenesis in treated differentiated C2C12.

Statistics: 1-way ANOVA * p<0.05.

Example 4 In Vitro Screening Assay for Compounds Promoting AMP-ActivatedProtein Kinase (AMPK) Activity in a Prototypical Skeletal Muscle CellLine (C2C12 Myotubes)

AMPK acts as a metabolic master switch regulating several intracellularsystems including the cellular uptake of glucose, the β-oxidation offatty acids, and the biogenesis of glucose transporter 4 (GLUT4) andmitochondria. The energy-sensing capability of AMPK can be attributed toits ability to detect and react to fluctuations in the AMP:ATP ratiothat take place during rest and exercise (muscle stimulation). As anexample, during a bout of exercise, AMPK activity increases(phosphorylation of AMPK, P-AMPK) while the muscle cell experiencesmetabolic stress brought about by an extreme cellular demand for ATP.Upon activation (AMPK phosphorylation, P-AMPK), AMPK increases cellularenergy levels by inhibiting anabolic energy-consuming pathways (fattyacid synthesis, protein synthesis, etc.) and stimulatingenergy-producing, catabolic pathways (fatty acid oxidation, glucosetransport, etc.). Consequently, AMPK activation leads to an enhancementof mitochondrial function, including increased OXPHOS and mitochondrialbiogenesis.

C2C12 myoblasts were differentiated to myotubes by serum deprivation for4 days (Canto et al. (2009) Nature. 458:1056-60). Myotubes wereincubated for 1 hr with resveratrol (RSV), which served as a positivecontrol, or ellagic acid (EA) or urolithin A (UL) at a finalconcentration of 50 μM (all dissolved in DMSO, final concentration0.1%). DMSO was used as a control (final concentration of DMSO: 0.1%).At the end of the treatment, cells were washed 3 times with PBS, andAMP-activated protein kinase (AMPK) was assessed by Western blot.Following compound treatment, C2C12 cells were lysed in buffercontaining phosphatase inhibitors, and protein concentration wasdetermined using a standard Bradford assay. The equivalent of 25 μg ofprotein was used for separation on a 10% SDS-PAGE gel and subsequentlytransferred by standard Western blotting procedures. Antibodies directedto AMPK (Cell Signaling) and phosphorylated AMPK (P-AMPK, CellSignaling) were used for detection.

As depicted in FIG. SA, Western blot analysis for the phosphorylated,and thus the activated form of AMPK—i.e., P-AMPK, indicated that thelevels of phosphorylation of AMPK (P-AMPK) and hence activation of theAMP-activated protein kinase (AMPK) were indeed increased in cellstreated with ellagic acid or urolithin relative to control treatedcells. This data indicates that ellagic acid and urolithin A are bothAMPK activators. This further supports the observations that ellagicacid and urolithins induce an increase in mitochondria function.

Example 5 Screening Assay for Compounds Promoting Neurite Outgrowth inPC-12 Cells

Neurite outgrowth and the number of mean processes per cell in neuronalculture have been shown to correspond to neuronal function. Chronicstress has been shown to result in reductions both of dendritic lengthand branch number, an effect which was reversed upon the removal ofstress. Furthermore, it has been shown that this reversibility becameinhibited with aging (Bloss, Janssen et al. 2010). There is also furtherevidence that learning and novel sensory experiences are associated withincreases in spine formation and elimination of protracted processes.Hence synaptic structural plasticity plays an important role in learningand memory (Yang, Pan et al. 2009). Indeed, the level of neuriteoutgrowth and number of processes induced by compounds such as nervegrowth factor (NGF) correlates strongly with their neuroprotectiveabilities. With aging this synaptic plasticity becomes compromised andthere is an increased loss of spines and a decreased density of synapses(Dumitriu, Hao et al. 2010). Neurodegenerative diseases also have aneffect on neurite outgrowth. A-beta (Aβ) peptide, which plays animportant role in Alzheimer's disease, inhibited neurite outgrowth inmouse neuroblastoma cells. Therefore, by assaying the effects on neuriteoutgrowth in vitro, compounds and formulations with neuroprotectiveeffects on neurons under chronic stress, neurons undergoing aging, andneurons present in neurodegenerative diseases can be identified.

The in vitro effects on neurite outgrowth of the different ellagitanninsand their metabolites punicalagin (PA), punicalin (PB), tellimagrandin(TL), ellagic acid (EA), and urolithin (UA), were tested on cells of anoradrenergic rat pheochromocytoma cell line (PC-12 cells), which havebeen shown to differentiate in response to nerve growth factor (NGF)(Greene and Tischler 1976). Neuritic outgrowth in these differentiatedPC-12 cells has been shown to be strongly promoted by dibutyryl cyclicAMP (dbcAMP) (Gunning, Landreth et al. 1981), and this compound wasutilized as a positive control. As a negative control, the specificJanus N-terminal kinase (JNK) inhibitor SP600125, which has been shownto decrease different parameters of neurite outgrowth, was utilized(Xiao, Pradhan et al. 2006). The ellagitannins and their metabolitestested in the assays were synthesized or purchased from suppliers whichincluded Funakoshi, Sigma, and Chemos. Stock solutions were aliquotedand stored at −20° C.

PC-12 cells (ATCC CRL-1721) were cultured at 37° C., 5% CO2 in polyL-lysine-coated culture flasks in complete culture medium (RPMI 1640+10%heat-inactivated horse serum+5% fetal bovine serum).

Cell differentiation was performed in culture flasks 24 h after plating,in complete medium supplemented with 100 ng/mL NGF (2.5 S NGF,Invitrogen). The NGF-supplemented medium was renewed every third day,and differentiation was induced over an 8-day period.

All compounds to be tested were prepared just before the experiment as a50 mM stock solution in dimethylsulfoxide (DMSO). The final DMSOconcentration was 0.1% in the medium of all experimental groups.

For neurite outgrowth measurements, differentiated cells were washedwith phosphate-buffered saline (PBS), collected after detachment andreplated at a density of 5,000 cells/well (biocoat imaging 96 wellplate) in complete medium supplemented with 100 ng/mL NGF, with orwithout 10 μM SP600125 (negative control), 1 mM dbcAMP (positivecontrol), or tested compound at 5×10⁻⁷ M. In the undifferentiatedcontrol group, no NGF was added after replating.

After 72 h in culture, PC-12 cells were washed with PBS and fixed in 1%paraformaldehyde solution for 20 minutes. After 3 washes with PBS,immunofluorescence labeling was performed with Texas Red Maleimideprobe, which reacts with thiol groups of cysteine residues of proteins,permitting the visualization of the entire cell morphology, includingneurites.

Immunofluorescence analysis was performed in automated confocalmicroscopy. Images were acquired with a BD pathway 855 system, under ×20objective with 8×8 field montage. Neurite outgrowth was then measuredfrom acquired images with the neurite module of Metamorph® software.Total and mean outgrowth, total and mean number of processes per cell,and total and percentage of cells with extensive outgrowth (defined asoutgrowth longer than 20 μm) were analyzed.

All compounds, except PA and PB, increased the number of PC-12 cells inthe wells by >30%, as shown in FIG. 6, indicating a trophic effect forthese compound at a concentration of 0.5 μM (p<0.001 for UA, EA, and TLversus differentiated control (ctrl)).

Promotion of Neurite Outgrowth

As shown in FIG. 7 and FIG. 8, all compounds tested (PA, PB, TL, EA andUA) were able to induce robust neurite outgrowth from differentiatedPC-12 cells. The mean outgrowth (FIG. 7) showed an increase of >30% overdifferentiated control for all the compounds tested. The percent ofcells showing significant outgrowth (FIG. 8) was significantly greaterthan that observed for differentiated cells for all compounds tested(p<0.05 for UA and PB (26% increase), p<0.01 for PA (>26% increase),p<0.001 for EA and TL, (>37% increase)).

Promotion of Process Formation and Branching

The compounds PA, PB, UA, EA, and TL, all induced an increase in thenumber of processes when applied to differentiated PC-12 cells.Compounds (UA, p<0.05 (15.7% increase); PA, p<0.01 (26.3% increase); EAand TL, p<0.001 (>31% increase) were either as effective as or moreeffective than dbcAMP, the positive control, in promoting processformation (FIG. 9).

Neurite branching was significantly higher than that observed indifferentiated control, with most compounds inducing a two-fold increasein branching.

Example 6 Screening Assay for Compounds Promoting Neurite Outgrowth inPrimary Dopaminergic TH-Positive Neurons

Primary neurons due to their untransformed state, serve as a good invitro model for the effects of compounds on markers of neuronalplasticity and differentiation, such as neuronal outgrowth, andformation of dendrites and processes. The effects of differentellagitannin metabolites punicalagin (PA), urolithin (UA), ellagic acid(EA), and tellimagrandin (TL) on this process were examined. Thecompounds tested in the assays were purchased from suppliers whichincluded Funakoshi and Sigma, or were chemically synthesized. Stocksolutions were aliquoted and stored at −20° C.

Primary mesencephalic cultures were prepared from rat E14 embryos.Ventral mesencephalon was carefully dissected and dissociated. Cellswere then plated in DMEM F12 medium containing 10% heat inactivatedhorse serum at the density of 100,000 cells/well (96-well plate), withor without the JNK-specific inhibitor SP600125 (10 μM) (which served asa negative control) or dbcAMP (1 mM) (which served as a positivecontrol), or the compounds tested each at the doses of 0.1 μM.

72 h after plating, the effects on the neurite outgrowth of dopaminergictyrosine hydroxylase (TH)-positive neurons were measured from imagesacquired by automated confocal microscopy (X4 objective, montage 4×4)and covering the entire well surface, and quantified using the Neuriteoutgrowth module of Metamorph® software. Several representativeparameters of neurite outgrowth were thus generated: total and meanoutgrowth, total and mean number of processes per cell, and total andpercentage of cells with extensive outgrowth (defined as outgrowthlonger than 20 μm) were analyzed. All experiments were performed inquadruplicate.

Promotion of Neurite Outgrowth

As shown in FIGS. 10-16, compounds selected in the above PC-12 screeningassay were also able to induce neurite outgrowth in primarymesencephalic neurons at a concentration of 0.1 μM. Most compounds wereas effective as dbcAMP in promoting outgrowth per cell (>25% increase inoutgrowth) as measured by the mean outgrowth per cell shown in FIG. 10(p<0.001 for UA, GA, EA, TL vs control). All compounds tested performedas well or better than dbcAMP (FIG. 11).

Increase in Neurite Processes and Branching

All the compounds tested showed significant increases in the meanprocesses per cell (>10%) (FIG. 12), as well as in the maximum processlength (>10%) (FIG. 13).

Primary cells showed an increase in branching in the presence of thepositive control (dbcAMP). However, the JNK inhibitor SP600125 did notinhibit primary cell branching, as in PC12 cells, but was capable ofpromoting branching albeit not to the same level as dbcAMP (60% vs 86%increase seen for dbcAMP). The compounds UA, EA, and TL were able topromote branching to the same levels as dbcAMP (>111% increase inbranching, FIG. 14).

Increases in Dendrites Per Cell and Dendrite Length

Dendrite number was increased significantly for UA, EA, and TL, atlevels above that of dbcAMP, with all compounds showing an increase >18%(FIG. 15).

Ellagic acid, urolithin A, and tellimagrandin all produced increases indendritic length >26%, higher than that observed for dbcAMP (FIG. 16).

Example 7 Pomegranate Extract, Punicalagin, Ellagic Acid, and UrolithinA Reduce Weight Gain and Fat Mass in Mice Fed a High Fat Diet

Male C57BL6/J mice were purchased from Charles River Laboratory(L′Arbresle, France) at the age of 7 weeks and acclimatized to theanimal facility for 2 weeks before initiation of experiments. Mice werehoused in groups of 5 in standard housing conditions, with a 12 hrlight-dark cycle and free access to food and water. Beginning at 9 weeksof age, mice were fed a high-fat diet (HFD) (60% kcal from fat; D12492;Research Diets Inc., New Brunswick, N.J., USA) for fourteen weeks. Bodyweight was monitored weekly.

Mice in different treatment groups were administered (i) urolithin Amixed with food (food admix) to reach a dosing of 55 mg/kg bodyweight/day (mkd); (ii) ellagic acid mixed with food (food admix) toreach a dosing of 75 mkd; (iii) punicalagin (gavage) to reach a dosingof 90 mkd; or (iv) pomegranate extract (PE) (gavage) to reach a dosingof 140 mkd of total polyphenols. A typical pomegranate extract used inthese experiments had the following composition: polyphenols, 140 mkd;punicalagin, 13.1 mkd; and ellagic acid, 13.2 mkd. For animals treatedby gavage, gavage was performed daily (7 days/week) between 8:00 and10:00 a.m.; compounds were mixed with saline solution (0.9% NaCl) andprovided at a final volume of 5 mL/kg of body weight. Mice in high-fatcontrol groups were fed with the same diet as the experimental animals.Mice in corresponding different control groups were administered eitherhigh-fat diet alone or high-fat diet plus daily gavage with vehicle(saline). Another control group of mice were fed standard chow dietalone.

Body composition was monitored by EchoMRI (Echo Medical Systems,Houston, Tex., USA) 5 weeks after the initiation of the treatment forhigh-fat diet fed mice and 2 weeks after the initiation of the treatmentfor chow diet fed mice. Animals were placed individually into a plasticcylinder and then introduced into an EchoMRI system for about 2 min forbody composition scanning (lean and fat mass).

Results are shown in FIGS. 17A-17C and 18A-18B.

Mice fed with a high-fat diet (HFD) developed a severe obesity comparedto control mice fed with a standard chow diet (CD) (FIG. 17A). Bodyweight gain in untreated high-fat fed mice was associated with anincrease in the percentage of fat mass (FIG. 17B) and a decrease in thepercentage of muscle mass (lean mass) (FIG. 17C), both as measured byEchoMRI after 5 weeks of treatment. In mice fed a high-fat diet,treatment with urolithin A (administered by food admix) or withpunicalagin or pomegranate extract (PE) (both administered by gavage)prevented the onset of obesity with a strong reduction of body weightgain in treated HFD-fed mice compared to control HFD-fed mice (FIG.17A). Along with this, fat mass was significantly reduced in HFD-fedmice treated with urolithin A, punicalagin, or PE compared to untreatedHFD fed mice (FIG. 17B).

Mice fed a standard-chow diet and treated with either ellagic acid orurolithin A also saw a reduction in fat mass with a concomitant increasein muscle (lean mass), illustrating that these treatments favor themanagement of weight and a lean or muscular physique (FIG. 18B).

Example 8 Pomegranate Extract, Punicalagin, Ellagic Acid, and UrolithinA Increase Muscle Mass in Normal and Obese Mice

Male C57BL6/J mice were grouped and treated as described in Example 7.

In both standard chow diet-fed mice and HFD-fed mice, treatment with PE,punicalagin, ellagic acid or urolithin A resulted in a statisticallysignificant increase in the percentage of lean mass. Mice fed a high-fatdiet and treated with urolithin A, punicalagin, or PE saw a reduction infat mass with a concomitant increase in muscle (lean mass) (FIGS. 17Band 17C). Mice fed a chow diet and treated with either ellagic acid orurolithin A also saw a reduction in fat mass with a concomitant increasein muscle (lean mass), illustrating that these treatments favor themanagement of weight and a lean or muscular physique (FIGS. 18A and18B). Since lean mass is predominantly represented by muscle mass, theseresults illustrate how treatments with either PE, punicalagin, ellagicacid or urolithin A result in an increase in the proportion of musclemass in both normal and obese mice with respect to total body mass. Thiseffect was observed after as little as two weeks of treatment.

Example 9 Pomegranate Extract, Punicalagin, Ellagic Acid, and UrolithinA Increase Energy Expenditure in Normal and Obese Mice

Male C57BL6/J mice were grouped and treated as described in Example 7.In addition, however, basal energy expenditure of mice was measured byindirect calorimetry oxygen consumption, carbon dioxide production, andrespiratory exchange ratio), 8 weeks after the initiation of treatmentfor the HFD-fed mice and 2 weeks after the initiation of treatment forstandard show diet-fed mice, using the Comprehensive Laboratory AnimalMonitoring System (CLAMS; Columbus Instruments, Columbus, Ohio, USA).Animals were first acclimatized for 22 h to CLAMS cages (roomtemperature 22° C.±1° C.) starting between 11 to 12 am. Then measurementwas performed for at least 20 h in the same condition. Measurementincluded an entire dark cycle. Parameters measured during CLAMS were thefollowing: (i) Oxygen consumption (VO₂ in mL/kg/h): VO₂ is directlycorrelated to energy expenditure; (ii) Carbon dioxide production (VCO₂in mL/kg/h); and (iii) Respiratory Exchange Ratio (RER): VCO₂/VO₂: RERis an indicator of the use of energy substrate. In a steady state, RERis equivalent to the Respiratory Quotient (RQ). Pure carbohydrate usegives RER=1, whereas pure fat burning yields an RER=0.7. A mixed dietgives a RER=0.85.

Results are shown in FIGS. 19A-19B and 20A-20B.

Oxygen consumption is a physiological marker of mitochondrial activityand energy expenditure. Treatments with either PE, punicalagin, ellagicacid, or urolithin A significantly increased oxygen consumption in mice.Ellagic acid and urolithin A increased energy expenditure in standardchow-fed mice (FIGS. 19A and 19B). This effect was observed after aslittle as 2 weeks of treatment. Pomegranate extract (PE), punicalagin,and urolithin A treatment increased energy expenditure in HFD-fed mice(FIGS. 20A and 20B).

Example 10 Pomegranate Extract, Punicalagin, Ellagic Acid, and UrolithinA Increase Use of Fatty Acids as Energy Substrates in Normal and ObeseMice

Male C57BL6/J mice were grouped and treated as described in Example 9.

As stated above, in addition to oxygen consumption, indirect calorimetryalso monitors carbon dioxide production. The ratio between carbondioxide production (VCO₂) and oxygen consumption (V02) is calledRespiratory Exchange Ratio (RER). RER is an excellent indicator of theuse of energy substrates. In a steady state, RER is equivalent to theRespiratory Quotient (RQ). A preferential use of carbohydrates as energysubstrate gives a RER close to 1, whereas a use of fat as energysubstrate (fat burning) yields a lower RER which is close to 0.7 whenfatty acids are preferentially used.

As depicted in FIG. 21A-21B and FIG. 22A-22B, PE, punicalagin, ellagicacid, and urolithin A treatment significantly decreased RER in both chowdiet and HFD-fed mice. This effect was dramatic in chow diet fed micetreated with ellagic acid and urolithin A (FIG. 21A-21B). These resultssupport the shifts in body composition observed following theconsumption of PE, punicalagin, ellagic acid or urolithin A which favora more muscular (lean) physique with reduced fat composition.

Example 11 Pomegranate Extract, Punicalagin, and Urolithin A DecreasePlasma Levels of Triglycerides and Free Fatty Acids in Obese Mice

Male C57BL6/J mice were grouped and treated as described in Example 7.In addition, plasma biochemistry was performed 14 weeks after theinitiation of the treatment using a standard automated clinicalchemistry analyzer (Dimension Xpand, SIEMENS). Animals were fasted for12 h (from 8 pm to 8 am) before blood collection. Approximately 500 μLof blood was collected from vena cava in anesthetized animals underisofluorane anesthesia. Blood was collected in heparinized tubes andimmediately placed on wet ice. Plasma was prepared by centrifugation(1500×g, 15 min, 4° C.). Plasma samples were then transferred in clean1.5 mL microtubes and stored at −80° C. until biochemical measurementswere performed on a standard automated clinical chemistry analyzer(Dimension Xpand, SIEMENS) using corresponding kits.

Circulating levels of triglycerides and free fatty acids was measured bystandard biochemistry in the blood of control and treated HFD-fed mice(FIG. 23A-23B). PE, punicalagin and urolithin A treatment led to astatistically significant improvement of plasma levels of triglyceridesand free fatty acids. These results indicate that PE, punicalagin, andurolithin A are effective in treating dyslipidemia in obese mice andconsequently can act to improve cardiovascular function and preventcardiovascular disease.

Example 12 Punicalagin, Ellagic Acid, and Urolithin A Improve GlucoseTolerance in Obese Mice

Male C57BL6/J mice were grouped and treated as described in Example 7.In addition, glucose tolerance test (GTT) was performed for HFD-fed micewhich developed glucose intolerance. Glucose tolerance test wasmonitored 10 weeks after the initiation of the treatment by Oral GlucoseTolerance Test (oGTT). Animals were fasted for 12 h (from 8 pm to 8 am)before oGTT. The day of the oGTT, a small drop of blood (<2 μL) wascollected from the lateral tail vein and glycemia was monitored using aglucometer (AccuCheck Aviva, Roche Diagnosis). Each animal thenreceived, at time 0, an oral dose of D-glucose at a dosing of 2 g/kgbody weight. Glycemia was then monitored at times 15, 30, 45, 60, 90,120, and 150 min after oral glucose load.

As in humans, high fat diet feeding in mice resulted in the onset ofobesity and type 2 diabetes which is characterized by a severe glucoseintolerance as assessed by the follow-up of glycemia immediatelyfollowing an exposure to glucose (2 g/kg of body weight) (Glucosetolerance test) (FIG. 24A-24B). As depicted FIG. 24A-24B, punicalagin,ellagic acid, and urolithin A treatment improved glucose tolerance inHFD-fed mice. Consequently, these treatments may also be effectivetherapeutic approaches for the treatment of type 2 diabetes.

Example 13 Urolithin A Increases Mitochondrial Function in Aged C.elegans

C. elegans strains were cultured at 20° C. on nematode growth media(NGM) agar plates seeded with E. coli strain OP50. Strain used waswild-type Bristol N2 provided by the Caenorhabditis Genetics Center(University of Minnesota). Urolithin A was dissolved in DMSO. Animalswere exposed to compounds from eggs on plates seeded with live OP50bacteria. Control plates were prepared with the correspondingconcentration of DMSO (0.1%).

Measurement of oxygen consumption is a direct indicator of mitochondrialactivity. The effect of urolithin A on mitochondrial activity in aged C.elegans (10 days old) was assessed by treating C. elegans with urolithinA for 10 days of adulthood, at which time oxygen consumption wasmeasured using the Seahorse XF24 equipment (Seahorse Bioscience Inc.,North Billerica, Mass.). 250 ten-day-old C. elegans were used percondition. C. elegans were recovered from NGM plates with M9 medium,washed three times in 2 mL M9 to eliminate residual bacteria, andresuspended in 500 μL M9 medium. Worms were transferred into 24-wellstandard Seahorse plates (#100777-004) (50 worms per well) and oxygenconsumption was measured. The basal oxygen consumption of the worms wasfirst measured over 30 minutes at 5 minute intervals (0 min, 5 min, 15min, 20 min, 25 min and 30 min) with 5 replicates per interval.Respiration rates were normalized to the exact number of worms per welldetermined after the completion of the experiment using astereomicroscope. After determining the basal oxygen consumption,uncoupled oxygen consumption was measured by addingcarbonylcyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) at the 30minute time point to the media in order assess the maximal oxygenconsumption capacity and maximal mitochondrial capacity. Uncoupledoxygen consumption was measured at 5 minute intervals (35 min, 40 min,45 min, 50 min, 55 min and 60 min) to permit measuring the mitochondrialfunction over time. FCCP is a chemical uncoupling agent that abolishesthe obligatory linkage between the respiratory chain and thephosphorylation system which is observed with intact mitochondria. Thiseffect is due to the amphipathic properties of the molecule whichdissolves in mitochondrial phospholipid bilayers. This dramaticallyincreases ionic permeability of the mitochondrial membrane and generatesdramatic proton leak leading to increase in oxygen consumption due tothe quenching by oxygen of the electrons pumped into the respiratorychain in parallel to the proton leak. Since this oxygen consumption isdissociated (uncoupled) to ATP production (oxidative phosphorylation),FCCP increases oxygen consumption while decreasing the generation ofenergy (ATP) by the mitochondria. Fully uncoupled mitochondria, asachieved with FCCP, display the maximal capacity of their mitochondrialrespiratory chain (maximal oxygen consumption) without the “brake” thatoxidative phosphorylation and energy production represents).

The results f that urolithin A increases the maximal mitochondrialcapacity of aged C. elegans, as depicted by a prolonged effect onincreased uncoupled respiration in worms treated with urolithin A versuscontrol (DMSO) treated worms. Control, untreated worms showed a briefincrease in uncoupled respiration which quickly returned to basal levelsof oxygen consumption. Urolithin A-treated worms showed a more extendedelevation in oxygen consumption. The extent of enhanced mitochondrialactivity is shown by comparing the area under the curves (AUC) duringthe decoupling period with the average coupled respiration employed asthe baseline. It was observed that urolithin A significantly increaseduncoupled respiration in aged worms as compared to control untreatedworms over the 30 minute period evaluated.

Example 14 Urolithin A Increases Mitochondrial Activity in C. elegans

C. elegans strains were cultured at 20° C. on nematode growth media agarplates seeded with HT115 bacteria and containing 50 μM urolithin A or acorresponding concentration of DMSO as a control. The worms were treatedfor 24 hours. The strains used were the SJ4103(zcIs14[myo-3::GFP(mit)]), which is a stable transgenic line expressinga mitochondrially localized green fluorescent protein (GFP) with acleavable mitochondrial import signal peptide under the control of thespecific body wall muscle promoter myo-3. GFP expression andquantification was carried out according to the protocol previouslydescribed (Durieux et al., 2011). Worms were treated with 50 μMurolithin A from eggs, and GFP was monitored after one day of adulthood.Fluorimetric assays were performed using a Victor ×4 multilabel platereader (Perkin-Elmer Life Science). Eighty worms were picked at random(20 worms per well of a black-walled 96-well plate) and each well wasread four times and averaged.

The results in FIG. 26 show that treatment of worms with urolithin Ainduced the expression of the mitochondrial GFP-reporter driven by themuscle-specific myo-3 promoter in C. elegans. This striking increase inGFP expression provides clear evidence that mitochondrial capacityincreased due to the urolithin A. To permit such an increase in observedGFP signal, mitochondria in muscle must either be enlarged or morenumerous in these worms.

Example 15 Effects of Pomegranate-Derived Compounds on Mood andCognition in Response to Chronic Stress

7-week-old C57BL/6J wild-type male mice were exposed to chronicunpredictable stress for a period of four weeks. Several behavioralexperiments were carried out before, during, and after the chronicstress period to determine the impact on mood and cognition. As has beenpreviously been reported, chronic stress negatively impacts mood andcognition. Natural compounds derived from pomegranate were administeredto these mice to determine what impact these compounds would have onameliorating this negative impact on mood and cognition.

Mice were habituated to our animal facility for 9 days before beginningthe experiments. All mice were housed in groups of three in standardplastic cages, and they were kept under a 12 h light/dark cycle (7:00a.m.-7:00 p.m.) with ad libitum access to food and water. All theprocedures carried out were performed in accordance with the SwissNational Institutional Guidelines on Animal Experimentation and wereapproved by the Swiss Cantonal Veterinary Office Committee for AnimalExperimentation.

Characterization of Animals

After adaptation to the animal facility, all mice were characterized interms of body weight, anxiety-like behavior in the elevated zero maze(EZM), and locomotion and exploration in the open field and novel objectassays. The objective of these experiments was to match animalsaccording to their anxiety and exploration rates in order to establishexperimental and control groups that are equivalent according to thesetraits.

Elevated Zero Maze

Anxiety was measured in an elevated zero maze (EZM). Mice were observedfor 5 min in the EZM (a 5.5-cm-wide annular runway with a diameter of 46cm and raised 46 cm above the ground) under dim and dispersed lightconditions. Two opposing 90° sectors were protected by 13.5 cm highinner and outer walls. Thus, three zones were defined as follows: anintermediate zone comprising four 30° segments at the ends of theprotection walls separated by the two 50° wide closed/protected and thetwo 70° wide open/unprotected exploration zones. With these boundaries,the entries into the open sectors were detected only when the animalentered into them with all four paws. The trajectories of each mousewere automatically recorded by video tracking (Ethovision 3.0, Noldus,Wageningen, Netherlands). The total number of entries into all thesectors served as an indicator of spontaneous locomotor activity, whiledifferences in the number of entries and the time spent in the opensectors was taken as indicators of anxiety. Between sessions the mazewas cleaned with 4% ethanol/water.

Open Field and Novel Object

Locomotion and reactivity to an open field (OF) was assessed in a whitequadratic box (50×50×37 cm) under dim and dispersed light conditions.The mouse is placed into the center of the field and allowed to movefreely during 10 min. The total distance moved, frequency of entries tothe center, time and percent time in the center of the OF were analyzed.Avoidance of the interior or “unprotected” area of the field isinterpreted as an anxiety-like behavior. Measures of total distance areused as an index of activity. Exploratory behavior was assessed by usingthe novel object (NO) test. The NO test was performed immediately afterthe OF test. A small, metallic object (3×1.5×5 cm) was placed into thecenter of the open field while the mouse was inside. Mice were giventhen 5 min to freely explore the novel object. The time spent in thecenter and the periphery of the compartment, number and latency ofentries to the center, and total distance moved in the center and thewhole compartment were analyzed. Percent time and distance the micespent in the center, exploring the novel object, were considered asindicators of “focused” exploratory activity.

Treatment with Pomegranate-Derived Extract

Three weeks before the initiation of the chronic stress protocol micewere separated into four different groups. One group received standardmouse chow diet (Control), while the remaining three groups receivedvarying doses of extract 1011, an extract derived from pomegranatejuice. Low dose corresponded to an extract dose of 21 mg/kg/d of gallicacid equivalents of polyphenols (GAE PPE), the medium dose correspondedto an extract dose of 43 mg/kg/d of GAE PPE, the high dose correspondedto an extract dose of 86 mg/kg/d of GAE PPE (see Table 5).

TABLE 5 Pomegranate Powder Extract 1011. Extract 1011 PolyphenolsPunicalagin Punicalin Ellagic Acid Low Dose 21 mg/kg/d 2.1 mg/kg/d  5.2mg/kg/d 2.0 mg/kg/d Medium Dose 43 mg/kg/d 4.2 mg/kg/d 10.5 mg/kg/d 3.9mg/kg/d High Dose 86 mg/kg/d 8.5 mg/kg/d  21 mg/kg/d 7.8 mg/kg/d

Treatment with the diet began three weeks before the initiation of thechronic stress protocol and continued until the termination of theexperiment.

Treatment with Urolithin A, a Pomegranate-Derived Metabolite

Three weeks before the initiation of the chronic stress protocol, micewere separated into two groups. One group received standard mouse chowdiet (Control), while the other group received a diet containingurolithin A, delivered at a dose of 25 mg/kg/d.

Chronic Unpredictable Stress

The unpredictable chronic stress protocol involved exposing animals to adaily stressful situation at an unpredictable moment for 4 weeks(between 8 am and 4 pm, and randomly distributed over the 28 days). Thestress stimuli used were either: 6 min tail suspension; 3×0.4 mAinescapable foot-shock; 4 h exposure to soiled, damp sawdust; 2 hexposure on an elevated platform; 1 h immobilization in a plastic tube;30 min exposure to 16° C.; 2 days inversed light/dark cycle; 10 minexposure to an older, aggressive conspecific; intense light exposure(600 lux); 2 h overcrowded cage (6 mice) and 8 h with a 40° cageinclination. All animals were weighed and the state of their coat wasevaluated on a regular basis (every 3-5 days). During this experimentone group of mice was exposed to the chronic stress and the other groupof animals was left undisturbed and served as controls.

Behavioral Assays Tail Suspension Test

The tail suspension test (TST) is used as a model for assessingantidepressant-like activity in mice. The test is based on the fact thatanimals subjected to the short-term (6 min.), inescapable stress ofbeing suspended by their tail, will develop an immobile posture. Themouse was hung on a metal bar by an adhesive tape placed 20 mm from theextremity of its tail. The distance between the floor and the bar wasapproximately 25 cm. Immobility is defined as the absence of initiatedmovements and includes passive swaying. Investigation time, whichincluded immobility, struggling and climbing, was scored from videotape.

As shown in FIG. 27, chronic stress resulted in an increased immobilityin the TST, an indicator of increased depression and sense ofhelplessness. However, mice treated with increasing doses of pomegranateextract demonstrated a reversal in this pattern and a restoration ofmobility and struggling to levels observed in non-stressed mice. Thusthe pomegranate extract prevents the depression response observed inchronically stressed non-treated mice.

Contextual Recognition

Contextual fear conditioning is a measurement of an animal's ability toremember a particular context. In this assay, mice are placed in a boxand then receive two mild shocks, one minute apart. In reaction to theshocks mice freeze. The ability of mice to recognize the context inwhich they received the shock is tested by placing them back in the boxat a later timepoint. If they recognize the context, mice will freeze inanticipation of receiving a shock.

In normal mice, the ability to recognize the context in the absence ofany shocks is a measurement of contextual memory. Mice with bettercontextual memory recognize the initial context better and thus have ahigher level of freezing.

This assay can also be used to measure anxiety in stressed mice. Instressed mice, increased anxiousness could be observed in increasedfreezing reaction time in response to the initial shocks, as well as alonger extinction period for the memory of the context. Extinction ofthe contextual memory is measured by placing the mouse in the samecontext once a day for several days in the absence of the initialadverse stimulus. With time mice unlearn the association of the contextwith the adverse stimulus, which is evidenced by a gradual decrease infreezing. In stressed mice that are anxious this extinction of theadverse memory takes longer.

Contextual fear conditioning was used to test the effect of pomegranateextract on anxiety induction (i.e., learned anxiety) in mice in responseto contextual recognition. Training and testing took place in a rodentconditioning chamber (20×20×28 cm), placed into a plexiglass box andilluminated by a 20-W bulb. The side walls of the conditioning chamberwere constructed of white methacrylate, and the door and the top coverwere constructed of plexiglass. The floor consisted of 20 steel rodsthrough which a scrambled shock from a shock generator could bedelivered. Ventilation fans provided a background noise of 68 dB (wholesystem: Panlab, S.L., Barcelona, Spain). Fear conditioning to thecontext was performed during the third week of the chronic stressprotocol in the stressed group. On the day of fear conditioning, micewere transported from the colony room to the adjacent behaviorallaboratory and placed in the conditioning chamber. Training consisted ofexposure of the mice to a conditioning context for 3 min followed bythree electric foot-shocks (2 sec, 0.4 mA) delivered after every min.After the last foot-shock the animal stayed for 30 s in the chamber. Thefear conditioning chamber was thoroughly cleaned with 0.5% acetic acidbefore each mouse was placed into the box. To determine the effect ofchronic stress and the various doses of pomegranate extract on the levelof anxiety induced by this contextual memory, the level ofanxiety-induced behavior in response to this context was measured. Thefollowing behavioral responses, known to be sensitive to levels ofanxiety, were examined: % freezing, % rearing, and % grooming. Thesebehavioral measurements were performed 48 h later after re-exposing themice to the conditioning context for 8 min. After training and testingsessions, animals were immediately returned to their home cages.Animals' behavior was recorded and later scored with in-house-madebehavior observation software by an observer blind to the treatment ofthe animals.

Freezing, defined as a lack of movement except for heart beat andrespiration, was scored and used as an index of anxiety. Freezing timewas transformed to percentage freezing levels. Pomegranate extractshowed a dose-dependent response, with a significant reduction in the %freezing at the highest dose (FIG. 28), indicating a protection againstanxiety. Similar reduction in anxiety behavior is seen in rearing, witha significant and dose dependent protection of the rearing behavior uponadministration of pomegranate extract (FIG. 29). Completing theseobservations is the strong suppression of anxiety-induced inhibition ofgrooming by the highest dose of pomegranate extract (FIG. 30). Theseresults demonstrate that the pomegranate extract and compounds reducedexperience-induced anxiety in mice.

This decrease in experience-induced anxiety in chronically stressed micewas also observed for urolithin A, a metabolite of punicalagin. In thisstudy, levels of anxiety were measured by the extinction of memory ofthe adverse context provided in the contextual fear assay describedabove. In this study the mice that have undergone training using thecontextual fear paradigm are exposed to this context daily for four daysbut in the absence of any adverse stimulus. The ability to recognize thecontext is measured by freezing during a period of 3 minutes ofobservation. Increased levels of anxiety have been shown to lead to alonger period for extinction for the memory of an adverse context. Asshown in FIG. 31, mice that have undergone chronic stress showed aslower period of extinction than normal mice. However, upon treatmentwith urolithin A at a dose of 25 mg/kg/d, mice that had undergonechronic stress showed a significant improvement in adverse memoryextinction, demonstrating that urolithin A, like punicalagin, is able toreduce anxiety in mice that have undergone chronic stress.

Morris Water Maze

Spatial memory and learning is affected by chronic stress. The Morriswater maze apparatus consisted of a large white circular pool (140 cmdiameter) filled with opaque colored water (25° C.+/−1° C.) and with aplatform (10×10 cm²) submerged 1 cm under the water surface. The watermaze is surrounded by gray curtains (25 cm from the pool periphery)containing several prominent visual cues. Before testing the mouse istrained to learn the location of the platform. Utilizing the prominentvisual cues, the mouse learns to locate the platform. The learning phasebegins with a habituation phase in which mice are introduced to theroom, apparatus, and the water by giving them a 2-min free swim trialwith no platform present. Data is collected using a video camera fixedto the ceiling, connected to a video tracking system (Ethovision 3.0,Noldus, Wageningen, Netherlands).

Following a habituation session (day 0), mice were submitted todifferent protocols to sequentially assess their spatial learningabilities (days 1-3). Spatial learning sessions were conducted on threeconsecutive days (days 1-3), performing four trials per day with aninter-trial interval (ITI) of 6 min between each trial.

Each trial started by introducing the mouse into the maze with the aidof a cup, facing the pool wall, and at one of four possible positionsthat were randomly balanced between trials and days. The distancebetween the mouse and the platform was measured at each sampling time,with 25 sampling times collected per second. These distances were thensummed for the 60 second period, to give a measurement of the distanceto platform (cm) for each trial. If a mouse did not find the platformwithin 60 sec, it was gently guided toward it. Each mouse had to remainon the platform for 20 sec before it was returned into its waiting cage.

The results of this example demonstrated that chronic stress had asignificant negative impact on learning and spatial memory. During thetraining session there was a significant increase in the distancetraveled to reach the platform compared with non-stressed control,showing that chronic stress impairs the normal memory forming duringlearning (FIG. 32). Treatment of mice with pomegranate extract was ableto protect against these negative effects of chronic stress on learningand associated memory. A dose-dependent effect was observed in micereceiving the pomegranate extract, and treated, chronically stressedmice were able to perform at the same levels of non-stressed controls(FIG. 33).

A similar effect was observed for mice treated with urolithin A, asshown in FIG. 34. Mice having undergone the chronic stress protocolshowed erratic learning, as evidenced by the high variability betweensequential trials. Treatment of chronically stressed mice with urolithinA at a dose of 25 mg/kg/d showed a stabilization of this variability.This highlights the fact that urolithin A, a downstream metabolite ofpunicalagin, is also able to protect against these negative effects ofchronic stress on cognition, including learning and memory.

In summary, these results together demonstrate that pomegranate extractand derived compounds such as urolithin A are able to act to reduce thenegative impacts of chronic stress on cognition, including memory andlearning. Additionally, the pomegranate extract and derived compoundshave anti-depressive activity as seen in the tail suspension test, anddecrease anxiety caused by chronic stress. The results also demonstratethat pomegranate extract prevents the deterioration of memory andlearning performance and spatial recognition normally observed followingchronic stress.

Example 16 Effect on Memory and Cognition in the Aged Rat Model

During aging there are several effects on cognition and memory, whichcan be recapitulated in the rat model of aging. For a review seeGallagher and Rapp (1977) Annu Rev Psychol. 48:339-70. The aged ratmodel has been extensively used to characterize the effects of aging onmemory and cognition. In the experiments presented here, improvedperformance was observed in the presence of pomegranate extract.

Aged Sprague-Dawley rats (beginning at 19 months old) receivedpomegranate extract (1108) in their drinking water, at a concentrationof 0.34 mg/mL polyphenols (PPE). Polyphenols content were measured usingthe Folin-Ciocalteu spectrophotometric method, with the phenolic contentexpressed as gallic acid equivalents. Control treatment consisted of1.36% sucrose, 0.12% D-glucose, and 0.12% D-fructose dissolved in water.The rats on average consumed 30 mL/day of both the control and 1108treatments (see Table 6), with an average weight of 660 g/rat. Thisresulted in a dose of 15 mg PPE/kg/d or 1.1 mg punicalagin/kg/d foranimals receiving the 1108 extract.

TABLE 6 Pomegranate liquid extract. Extract 1108 Polyphenols  15 mg/kg/ddose delivered Punicalagin 1.11 mg/kg/d dose delivered

After 2.5 months of treatment, short term working memory was evaluatedusing a social recognition task, a standard test involving socialcognition. Thor and Halloway (1981) Animal Learning Behavior. 9:561-5.In this task, each aged rat was placed in its home cage together with ajuvenile male Sprague-Dawley rat (<5 weeks old) for 5 minutes. Thirtyminutes later, the same exact procedure was repeated with the samejuvenile to determine a second time the degree of interaction betweenthe two animals. Less contact is expected in the second interaction, asthe two animals have had a previous interaction. This decrease incontact between the animals is a measure of cognitive performance andmemory retention. Thirty minutes later, a novel juvenile rat was placedfor 5 minutes together with the aged rat, in order to measure whetherthe animal can discriminate between the two different juvenileindividuals. During each period of contact between the two animals, thetotal time of contact was measured to assess the intensity of socialinteraction.

Results are shown in FIG. 35. Control-treated aged animals showed nopreference for the familiar object and spent equal time exploring bothobjects, an effect that has been previously shown in aged rats andthought to reflect a decline in temporal order memory during aging.Hauser et al. (2009) Behav Neurosci. 123:1339-45. However, rats treatedwith extract 1108 showed a decrease in the time spent with the samejuvenile during the second exposure period, as well as an increase inthe time of interaction with a novel juvenile rat. This observeddifference illustrates the protective benefit of extract 1108 on memorydevelopment and retention.

Example 17 Effect on Spatial Memory in Aged Rat Model

Spatial memory also has been reported to be affected by aging, with adecline in performance resulting from aging. Bergado et al. (e-pub Oct.29, 2010) Spatial and emotional memory in aged rats: a behavioralanalysis. Neuroscience. To examine the effects of pomegranate extract onspatial memory decline during aging, aged Sprague-Dawley rats (beginningat 19 months old) were treated with pomegranate extract 1108 or controlin their drinking water as described for Example 16.

Aged rats were treated with the extract 1108 or an isocaloric controlfor three months, after which their learning and memory performance wereevaluated using the Morris water maze task, described in Example 15.

The learning abilities of each animal were evaluated through theirperformance on the reversal task (three trials). In this task theanimals were first taught the location of the platform in quadrant(WEST) through three training trials. The location of the platform isthen changed and it is placed in the opposite quadrant (EAST). Theanimals underwent three new training sessions to learn the new locationof the platform. The effort to determine the new location of theplatform, as measured by the distance traveled before locating theplatform, was measured. Results are shown in FIG. 36. Animals treatedwith the extract were significantly more efficient at localizing theplatform in the reversal test (one-way ANOVA, P<0.02; control N=11; PJ:N=13; extract: N=14), demonstrating a therapeutic benefit of theadministered extract for this aspect of spatial memory.

Example 18 Effects of Pomegranate-Derived Compounds on Spatial andWorking Memory in Alzheimer's Disease

Alzheimer's Disease (AD) has been shown to have detrimental effects onspatial memory, an effect that is also observed in AD mouse models ofthe disease. To determine the effects of pomegranate-derived compoundson ameliorating spatial and working memory in AD, various pomegranateextracts and punicalagin were tested in two behavioral assays of spatialmemory, the Y maze and the Morris water maze.

Y Maze

In this study, the 5XFAD mouse model of AD was utilized. The 5XFAD mousemodel for Alzheimer's disease is based on genetic modifications(introduction of the mutated human APP and PS1 genes) leading to theproduction of amyloid β peptide (Aβ) in brain tissue. These mice werefound to have a significant decline in cognitive performance in the Ymaze as early as 7 months of age.

To determine the effect of pomegranate-derived compounds, a pomegranateextract (PE) derived from whole pomegranate was delivered by gavage at adose of 60 mg/kg/d of polyphenols, which includes approximately 5.6mg/kg/d of punicalagin. Mice were gavaged 3 times weekly beginning at 3months of age until the end of treatment. Mice were tested after 7months of age for the effects of PE on working memory on the Y maze.Mice were placed in the Y maze for 15 minutes and were allowed toexplore two arms, the third arm being closed. Four hours later, theanimal was placed again in the maze for five minutes, this time with thethird arm open, allowing the mouse to have the possibility to explorefreely all three arms. Exploration activity in the novel arm assessedthe ability of the animal to recognize that this particular zone has notbeen explored yet, according to spatial clues. Mice were scored asmaking a correct alteration during exploration if they explored each ofthe three arms, and not just the first two presented.

As shown in FIG. 37, a significant improvement in working memoryperformance, as measured by the number of correct alterations, wasobserved in SXFAD mice treated with PE.

Morris Water Maze

The pomegranate extracts 31008, 61109 and 71109 were tested in a secondtransgenic animal model of Alzheimer's disease expressing both theamyloid mutant London mutations and the prenisilin-1 human mutation.Animals in this model develop plaques by 4 months of age and memorydeficits by 6 months. Dense plaque load is visible after 7 months.

In one set of experiments, four-month-old APP-PS1 transgenic mice werefed with a fixed dose of approximately 97 mg total polyphenols/kg/day,which includes approximately 15 mg/kg/d of punicalagin of the extract31008, which was derived from whole pomegranate, via their drinkingwater. In one set of experiments, four-month-old APP-PS1 transgenic micewere fed with a fixed dose of approximately 468 mg total mg/kg/day ofthe extract 61109, which was highly enriched for punicalagin (>91%), viadrinking water. In one set of experiments, four-month-old APP-PS1transgenic mice were fed with a fixed dose of approximately 180 mg totalpolyphenols/kg/day of the extract 71109, which was derived frompomegranate husk, via drinking water. After 3 months of feeding, themice (then 7 months old) were tested in the Morris water maze spatialtest.

The Morris water maze was performed during days 84-87 of treatment. Thepool (a white, circular vessel 1 m in diameter) contained water at 20°C. with titanium-dioxide as an odorless, nontoxic additive to hide theescape platform (1 cm beneath the water level). Swimming of each mousewas videotaped and analyzed (Ethovision, Noldus information Technology,Wageningen, Netherlands). Prior to training, each mouse was placed ontop of the platform for 15 seconds. For place navigation tests, micewere trained to locate the hidden platform in five blocks of threetrials over three consecutive days. Each trial consists of a forced swimtest of maximum 120 seconds, followed by 60 seconds of rest. The timeeach mouse needed to locate the platform was measured during the fiveconsecutive blocks of training to determine a learning curve for eachmouse.

Twenty-four hours after the final training, each animal underwent aprobe trial with the platform removed. Mice were allowed to search forthe missing platform for 60 seconds and the search time spent in eachquadrant of the pool, as well as the number of crossings of the originalplatform position was measured. As shown in FIG. 38, the mice fed withExtract 31008 showed an increase performance in the probe test asdemonstrated by the increased frequency of crossings of the area werethe platform was formally located. Mice fed with extracts 61109 and71109 had even better performance.

The compositions of the extracts 61109 used in this experiment are shownin Table 7.

TABLE 7 Extract 61109 Punicalagin 91.3% (w/w) Punicalagin 295 mg/kg/ddose delivered

Example 19 Effects of Pomegranate-Derived Compounds on Depression,Anxiety and Cognition in Response to Early-Life Stress

Pomegranate-derived compounds were assessed for their ability to improvebrain functions, including cognition, depression and anxiety, in anearly-life stress model associated with maternal separation.

Early life stress has a significant impact on cognitive performance inlater adult life, including (i) increasing abnormal decision making andexcessive risk-taking; (ii) susceptibility to increased rates ofdepression and anxiety; and (iii) impaired learning and memory.

All procedures carried out were performed in accordance with the SwissNational Institutional Guidelines on Animal Experimentation and wereapproved by the Swiss Cantonal Veterinary Office Committee for AnimalExperimentation.

Early Life Stress Produced by Maternal Separation

At postnatal day 1, pups were culled to have 6 pups per mother. Frompostnatal day 1 to 14, unpredictable maternal separation (MS) of aperiod of 3 hours daily was carried out. Maternal separation wasperformed at random times (from 8 am to 2 pm) to avoid the habituationof the mother to the procedure. The protocol consisted of removing pupsfrom their mother to another cage at room temperature for a 3 hourperiod, after which the pups were returned to their original nest. Thesegroups are denoted as early-life stress in the figures. A control groupof dam/pups was left undisturbed and is denoted in the figures asnormal.

Treatment with Punicalagin Isolated from the Pomegranate

One week after maternal separation mice were separated into two groups.One control group received the standard mouse chow diet (Untreated),while the other group received the ellagitannin punicalagin admixed intothe food and designed to deliver a dose of 90 mg/kg/day to the mice.Treatment with the diet began 1 week after the termination of thematernal separation treatment.

Behavioral Assays

The effects of early-life stress on depression, anxiety and cognitionwere examined utilizing the following behavioral assays which werecarried out 166 days after the completion of the maternal separationprotocol. Normally raised mice were compared versus maternally separatedmice (early-life stress) and maternally separated mice treated withpunicalagin.

Dark/Light Box Test

In this assay mice are placed in a PVC box which is separated into twocompartments: a dark compartment (15×20×25 cm, black PVC, and coveredabove) and a lit compartment (30×20×25 cm, white PVC and illuminated at200 lux), both linked by an interconnecting door (5×5 cm) (Ligna, Paris,France). The experiment is started by placing the animal in the darkcompartment, after which a camera records for a 5 minute period theamount of time the mouse spends in the lit area, the number oftransitions from the dark to lit area, and the latency to escape fromdark to lit area.

Normally, mice will avoid the illuminated area in the box. Maternallyseparated mice spent an abnormally long time in the illuminatedcompartment as compared to their non-maternally separated littermates asa consequence of their early-life stress (FIG. 39). This increase intime spent exploring the lit area reflects an impaired decision-makingbehavior characterized by abnormal and excessive risk-taking.

Punicalagin treatment of maternally separated mice reversed andnormalized the excessive risk-taking behavior observed and restored thedecision making process to normal (FIG. 39).

Elevated O-Maze (EOM)

Another behavioral assay that measures abnormal risk taking is theelevated O maze (EOM). In this assay an apparatus consisting of a ringwith a diameter of 41.5/46.5 cm (internal/external diameter) is dividedinto four equal parts. Two parts of the ring, opposite each other, areenclosed by walls that are 5 cm high. The remaining two parts of thering have no walls. The maze is elevated 1 m above the floor. Thenatural tendency of mice is to avoid open surfaces and spend more timein the enclosed regions of the ring that have the 5 cm walls, as opposedto the open regions of the ring.

To examine the effect of early life stress, mice were placed at theentrance of one of the areas of the maze with the 5 cm walls, with thenose facing the closed arm, and were allowed to explore the EOM for 5min. During this period animal behavior was videotaped. The time spentin each arm (closed versus open) was calculated, with an entry into thearm only being considered to have occurred when the animal placed allfour paws in the arm.

Normally, mice placed in the elevated O-maze will avoid the open regionsof the ring and spend limited time exploring this area. Mice stressed bymaternal separation spent an abnormally long time in the open sectionsof the O-maze as compared to their non-stressed littermates (FIG. 40).As also observed in the dark/light box test, this reflects an impaireddecision-making behavior in early-life stressed mice which ischaracterized by abnormal and excessive risk-taking.

Punicalagin treatment of maternally separated mice reversed andnormalized their abnormal excessive risk taking behaviour due toearly-life stress (FIG. 40).

Forced Swim Test

The Porsolt or Forced Swim Test is commonly used to test antidepressanttreatments (Porsolt et al., 1977a; Porsolt et al., 1977b). For thisbehavioral test, a mouse is placed in a 5 L cylinder (11 cm diameter and25 cm height) filled two-thirds full with water at 23° C. Animals wereconsidered to be engaged in swimming and mobile if there was a cleardisplacement of the body. Animals floating with minimal movement duringthe analysis period were considered to be immobile. Animal behavior wasrecorded over a 6-minute test period using a camera and a mirror behindthe cylinder. The first 2 minutes and the last 4 minutes of the swimmingwere analyzed separately for mouse swimming activity. Increasing levelof depression is correlated to an increase in mouse immobility,particularly during the last 4 minutes. Animals that have undergoneearly-life stress showed a significant increase in immobility ascompared to their non-stressed littermates, indicating an elevated levelof depression (FIG. 41). Punicalagin treatment of early-life stressedmice reversed this abnormal behavior (increased immobility) andincreased swimming activity to levels seen in non-stressed mice. Thisbehavioral effect of punicalagin demonstrates its activity as ananti-depressant (FIG. 41).

Contextual Fear Conditioning

Contextual fear conditioning was used to determine the effects of theellagitannin punicalagin on the susceptibility of adult animals,subjected to early life stress, to anxiety. Animals were trained in afear conditioning chamber (Context A, W x L x H: 30 cm×24 cm×26 cm)(PanLab) that contained a grid floor with stainless steel rods and wasconnected to a shock generator developed by Panlab. During training,animals were placed into the chamber one at a time. After four minutesof exploration inside the chamber, one foot shock (2 seconds and 0.4 mA)was administered, followed by a second foot shock (2 seconds and 0.4 mA)one minute later. Thirty seconds after the second foot shock, the mousewas placed back into its home cage. Animal behavior was monitored every2 seconds throughout the duration of the experiment. The period thatmice spent immobile in the chamber was considered as “freezing” and wasscored throughout these periods. The time the mice spent immobile afterthe first shock was recorded for a 60 second period and expressed as apercentage.

The behavior of mice to rest immobile and “freeze” in response to a footshock is a measure of their level of anxiety. The longer the durationthe “freezing” lasts during this behavioral test, the higher theanimals' level of anxiety.

Differences in freezing between the groups tested (normal non-stressed,early-life stress, and early-life stress+punicalagin) were observedafter the first shock (FIG. 42). Early-life stress led to increasedanxiety in mice as evidenced by the increased time spent freezingfollowing the foot shock, as compared to their non-stressed littermates(FIG. 42). Punicalagin treatment decreased and normalized these elevatedanxiety levels resulting from early-life stress, as shown by a reducedfreezing time following the foot shock (FIG. 42). These observations inthe early-stress model illustrate the anxiolytic effects of punicalagin.

The increased anxiety experienced by animals exposed to early lifestress is also observed in the extinction (i.e., disappearance) of thecontextual memory (i.e., the memory associating the context of theenvironment to the shock) induced by this assay. To examine the strengthof the anxiety developed during the contextual fear conditioning (asdescribed above), animals were placed into the same chamber and contextfor 3 min daily (same time of day, however no shock this time) for the12 days that followed the initial testing. Freezing behavior induced bythe simple recognition by the animals of the chamber, where theyreceived the initial shock, was measured during each of these daily3-minute periods.

Animal groups (normal non-stressed, early-life stress, and early-lifestress+punicalagin) showed differences in the decline of theircontextual recall of the shock over the subsequent 12 days (FIG. 43). Inthis graph, the duration of freezing is presented as the percent of timespent immobile on day 1 (for example, if a mouse was immobile for 60seconds on day 1 and 30 seconds on day 8, percent immobility is 100% onday 1 and is 50% on day 8).

Normal, non-stressed mice showed a predictable decline in contextualrecall during the 12 day period (FIG. 43). Early-life stressed mice hada heightened contextual recall, which is illustrated by a higher levelof freezing than their non-stressed littermates (FIG. 43). This shows aprolonged elevated level of anxiety in these maternally separated mice.Punicalagin treatment of early-life stressed mice had a clear impact onreducing anxiety as seen by the extinction of the contextual recall.Treated early-life stressed mice showed a faster extinction thanuntreated early-life stressed mice, characterized by reduced period oftime spent freezing between days 8 and 12 (FIG. 43).

Rotarod

To measure the effects of pomegranate-derived compounds on the negativecognitive impacts of maternal separation, effects on motor learning wereassayed using the rotarod behavioral assay. The rotarod apparatusconsists of a rod of 2 cm diameter. A mouse is placed on a rotating rodwhich is started at an initial speed of 5 rpm. The rod speed isgradually accelerated at a rate of 8 rpm/min until reaching a speed of45 rpm. The latency to fall was measured with a cutoff time of 300 sec.As shown in FIG. 44, mice that have undergone early-life stress sufferedfrom impaired motor learning. Maternally separated mice fell off therotarod faster than normal non-stressed mice. Treatment with punicalaginrestored motor learning skills in early-life stressed animals toperformance levels observed in normal non-stressed littermates.

Morris Water Maze

The Morris water maze behavioral assay was employed to assess thecognitive impact of maternal separation. In this assay, cognitivelearning is measured by the ability of a mouse to locate a hiddenplatform in a pool of opaque water. The apparatus consists of a pool(140 cm diameter) filled with water at 22° C. Mice escape from the waterby swimming to a hidden circular platform (15 cm diameter) placed 1 cmunder the surface of the water. By using visual cues located outside ofthe maze, mice are able to locate the platform and recall its locationduring subsequent trials. During the training phase, mice were placed attwo starting positions (alternating) every hour. The Morris water mazetask was performed with 8 trials at T1, 6 trials at T2, and 4 trials atT3 (on days 1, 2 and 3). Mice had a maximum of 60 sec to reach theplatform. Escape latency to reach the platform was measured by a videotracking system. As can be observed in FIG. 45, early-life stress had asignificant impact on cognitive learning, with mice taking a longerperiod of time to learn the location of the hidden platform, as shown bythe increased escape latency versus normal non-stressed mice. Treatmentof these maternally-separated mice with punicalagin reversed thisnegative impact of early-life stress, reducing the time to learn thelocation of the hidden platform to levels observed in normalnon-stressed mice. These results demonstrate the ability of punicalaginto reverse the long-term negative cognitive impacts of early-life stresson learning and memory formation.

Pomegranate-Derived Compounds

Taken together the data above demonstrate that compounds derived fromellagitannins are able to reverse to long-term negative impact ofearly-life separation on depression, anxiety, and cognition.

Example 20 Effects of Pomegranate-Derived Compounds on Memory andCognition in Normal Mice

Treatment with Pomegranate-Derived Compounds

Beginning at 3 months of age, mice were either fed (i) a standardcontrol diet such as AIN-93G; (ii) a diet containing punicalagin at aconcentration of 0.87 mg/kg, so as to deliver an approximate dose of 90mg/kg/day (for a period of 3 months); or (iii) a diet containingurolithin A at a concentration of 0.57 mg/kg, so as to deliver anapproximate dose of 55 mg/kg/day (for a period of 2.5 months). Actualdoses vary slightly depending on the food consumption of each individualmouse, as well as the weight of the mouse. Behavioral assessment ofcognition was measured after this period.

Behavioral Assay to Measure Effects of Pomegranate-Derived Compounds onCognition

To examine the effect of pomegranate-derived compounds on memory andcognition, mice were examined for improvements in contextual memoryutilizing the contextual fear conditioning assay. Mice were trained in afear conditioning chamber as described in Example 19.

During training, animals were placed into the chamber one at a time.After four minutes of exploration inside the chamber, one foot shock (2seconds and 0.4 mA) was administered, followed by a second foot shock (2seconds and 0.4 mA) one minute later. Thirty seconds after the secondfoot shock, the mouse was placed back into its home cage.

One day later, trained animals were returned to the chamber for a periodof three minutes. During this period mice were monitored for theirmovement. The amount of time spent immobile or “frozen” was scored as apercentage of the total time (3 minutes) under observation. The timespent immobile is a measure of the strength of the memory of the mice torecall the context in which they were trained. Treatment with both thepomegranate-derived ellagitannin punicalagin and the ellagic acidmetabolite urolithin A led to significant improvements in contextualmemory over untreated control mice as determined by their contextualmemory 24 hours following the training period (FIG. 46).

To determine the effect of these pomegranate-derived compounds on memoryretention, normal mice fed either (i) a control diet; (ii) punicalagin(for a period of 3 months) or (iii) urolithin A (for a period of 2.5months) were studied for their memory recall on days 1, 2, 3, 4 and 5after the initial contextual fear training.

Animals were placed in the same chamber and context for 3 min daily(same time of day, however no shock this time) for the 5 days thatfollowed the initial testing. Freezing behavior induced by the simplecontextual recognition by the animals of the chamber, where theyreceived the initial shock, was measured during each of these daily3-minute periods. The ability to recognize this environment in theabsence of the stimulus is a measurement of contextual memory.

With each passing day control untreated mice begin to have an extinctionof their memory for this contextual stimulus, as evidenced by a decreasein extent of freezing (FIG. 47). Mice treated with either punicalagin orurolithin A demonstrated an improved memory retention as compared tocontrol, untreated mice. This is illustrated by an ability to rememberthe initial context for a longer period, evidenced by a significantlylonger period for the extinction of the contextual memory (FIG. 47).

These results demonstrate that treatment with either punicalagin orurolithin A lead to improved cognition, as evidenced by a significantincrease in context recognition and improved memory retention.

Example 21 Effects of Pomegranate-Derived Compounds on Improving MusclePerformance in Normal Mice

Ellagitannin-derived compounds punicalagin and urolithin A wereevaluated for their ability to improve muscle performance. To examinethe benefits of punicalagin and urolithin A on improving muscleperformance, their effects were examined using two behavioral assays:(i) the rotarod assay, which measures muscle performance and motorskills, including coordination, and (ii) the treadmill endurance test,which measures muscle performance and endurance.

Behavioral Assays to Measure the Effects of Pomegranate-DerivedCompounds on Muscle Performance Rotarod Assay

Beginning at 3 months of age, mice were fed either a standard controldiet such as AIN-93G or a diet containing punicalagin to deliver a doseof 90 mg/kg/day for a period of 3 months.

To examine the effect of pomegranate derived compounds on muscleperformance and motor skills, mice were tested on the rotarod behavioralassay. The rotarod apparatus consists of a rod with a diameter of 2 cmwith 5 compartments, 5 cm wide. A mouse is placed on a rotating rodwhich is started at an initial speed of 5 rpm. The rod speed isgradually accelerated at a rate of 8 rpm/min. The latency to fall wasmeasured with a cutoff time of 300 seconds. Mice were tested for fourtrials. The latency to fall is a measure of the muscle performance andmotor skills of the mice, with a better performance reflected by alonger latency to fall. Both control untreated and punicalagin-treatedmice were tested. The ellagitannin punicalagin was able to significantlyimprove muscle performance and motor skills as compared to untreatedmice. Punicaligin-treated mice were able to remain on the rotarod for alonger time and at higher speeds compared to untreated mice duringsequential trial periods (FIG. 48).

Endurance Test

Normal 8-week-old mice were acclimated for 2 weeks prior to the start ofthe study. Mice were fed with a standard rodent diet (chow diet) or adiet containing urolithin A mixed with the food to reach a dosing of 55mg/kg/day delivered to the mice. Following 6 weeks of treatment, micewere tested for their muscle performance by means of an endurance test.

An endurance test was performed using a variable speed belt treadmillenclosed in a plexiglass chamber with a stimulus device consisting of ashock grid attached to the rear of the belt (Panlab, Barcelona, Spain).Mice were run at 10 cm/sec and a 0° of incline for 5 min. Speed was thenincremented by 2 cm/sec every 5 min, until mice were exhausted. Thedistance run and the number of shocks obtained over 5 min intervals wererecorded. Mice were considered exhausted and removed from the experimentwhen they received approximately 20 shocks in a period of 1 min. Controluntreated and urolithin A-treated mice were tested and compared fortheir performance.

Improved muscle performance and endurance is reflected by an ability torun at higher speeds on the treadmill. Mice will seek to avoid the shockand will run despite the increasing speed. At a certain point the miceare unable to keep up with the treadmill speed and are shocked. Afterreaching the threshold levels of shocks, mice are removed from thetreadmill. Mice having better muscle performance and improved endurancewill be able to keep up with the increasing speed of the treadmill andwill experience fewer shocks at a particular speed. Urolithin A-treatedmice ran at higher speeds than untreated control mice in this behavioralassay, illustrating that urolithin A improved muscle performance andendurance in this context (FIG. 49).

These results demonstrate that the ellagitannin punicalagin and itsmetabolite urolithin A are able to improve muscle performance and motorskills in mammals.

EQUIVALENTS

The invention has been described broadly and generically herein. Thoseof ordinary skill in the art will readily envision a variety of othermeans and/or structures for performing the functions and/or obtainingthe results and/or one or more of the advantages described herein, andeach of such variations and/or modifications is deemed to be within thescope of the present invention. More generally, those skilled in the artwill readily appreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present invention is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described andclaimed. The present invention is directed to each individual feature,system, article, material, kit, and/or method described herein. Inaddition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the scope of the present invention. Further, each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

INCORPORATION BY REFERENCE

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right physically to incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

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We claim:
 1. A method of treating or preventing a disease or conditionselected from the group consisting of a metabolic disorder, obesity,diabetes mellitus, a cardiovascular disease, hyperlipidemia,hypertriglyceridemia, elevated free fatty acids, and a metabolicsyndrome, comprising: administering to a subject in need thereof atherapeutically effective amount of a urolithin or a salt thereof. 2.The method of claim 1, wherein the disease or condition is a metabolicdisorder.
 3. The method of claim 1, wherein the disease or condition isobesity.
 4. The method of claim 1, wherein the disease or condition isdiabetes mellitus.
 5. The method of claim 1, wherein the disease orcondition is a cardiovascular disease.
 6. The method of claim 1, whereinthe disease or condition is hyperlipidemia.
 7. The method of claim 1,wherein the disease or condition is hypertriglyceridemia.
 8. The methodof claim 1, wherein the disease or condition is elevated free fattyacids.
 9. The method of claim 1, wherein the disease or condition is ametabolic syndrome.
 10. The method of claim 1, wherein the urolithin isurolithin A, urolithin B, urolithin C, urolithin D, or a combinationthereof.
 11. The method of claim 10, wherein the urolithin is urolithinA.
 12. The method of claim 10, wherein the urolithin is urolithin B. 13.The method of claim 1, wherein the subject is a mammal.
 14. The methodof claim 13, wherein the mammal is a human.
 15. The method of claim 1,wherein the urolithin is administered orally.
 16. The method of claim 1,wherein the urolithin is administered topically.
 17. The method of claim1, wherein the urolithin is administered parenterally.
 18. The method ofclaim 1, wherein the urolithin is administered as a food product, foodadditive, food ingredient, functional food, medical food, dietarysupplement, nutraceutical, nutritional supplement or oral preparation.19. The method of claim 1, wherein the urolithin is administered as apharmaceutical composition.